1. VALIDATION OF IN-HOUSE
CRYOPROTECTANT AND IN-
HOUSE DESIGN FOR
CRYOPRESERVATION OF DORSAL
ROOT GANGLION CELLS
Author:
Sarp Ertas
2172388
Supervisors:
Dr.Mathis Riehle
Dr. Jemma Roberts
A thesis submitted in partial fulfilment of the requirements for the
degree of
Master of Science in Biotechnology
School of Medical, Veterinary and Life Sciences
University of Glasgow
August 2016
2. 2
Abstract
The last two decades have seen a growing trend towards cryopreservation. in line with
the increasing need to be able to store and transport viable cells. As such a
considerable volume of literature has been published on different approaches
cryopreservation studies. These studies suggest that vitrification techniques yield
superior cell viability compared to other methods and can be achieved with simple
devices. This study was designed to develop a vitrification technique, which will later
be used for the transportation and storage of primary dorsal root ganglion (DRG)
neurons. In order to facilitate surface dependent vitrification, an in-house design
prototype was developed and optimized to provide the high-cooling rates required for
vitrification without immersing the cells directly in liquid nitrogen. Although this study
could be a cornerstone for further research, a clear benefit of the in-house device in
the prevention of ice formation, could not be identified in this analysis. The overall
structure of the study takes the form of three chapters, including confluence
determination, validation of the in-house CPA and surface dependent vitrification (the
in-house design).
Abbreviations
DRG: Dorsal root ganglion:
MeOH: Methanol
CPA: Cryoprotectant agent
CRF: Controlled rate freezer
QMC: Quartz Micro Capillary
DMEM: Dulbecco’s Modified Eagle
Medium
FBS: Foetal Bovine Serum
PBS: Phosphate Buffered Saline
DMSO: Dimethyl Sulfoxide
TRITC: Tetramethylrhodamine
FITC: Fluorescein Isothiocyanate,
DAPI: 4',6-Diamidino-2-Phenylindole
Calcein AM:Calcein acetoxymethyl ester
hTERT: Human telomerase reverse
transcriptase
NaCl: Sodium Chloride
MgCl2: Magnesium Chloride
BSA: bovine serum albumin
M: Molar
BBFM: Bambanker freezing media T-S: Trehalose supplemented
3. 3
Table of Figures
FIGURE 1: THE ILLUSTRATION OF THE EXTENDED PHASE DIAGRAM OF CELL PRESERVATION AT CRYOGENIC
TEMPERATURE. IN ADDITION, THE GRAPH ALSO DEMONSTRATES THE PHASE DIAGRAM OF EVAPORATIVE
DRYING, WHICH IS USED FOR DESICCATION OF THE CELLS IN THIS STUDY (HE, 2011). .............................................7
FIGURE 2 THE ILLUSTRATION OF THE IN-HOUSE DEVICE DESIGN (A). THE WORKFLOW DIAGRAM OF THE IN-HOUSE
DEVICE (B). THE ILLUSTRATION OF OPTIMIZED IN-HOUSE DEVICE TO ACHIEVE HIGH COOLING RATES AND IT IS
WORKFLOW. FOR THE CULTIVATION OF CELLS THE CENTRAL CIRCLE (BOROSILICATE GLASS) OF 35 MM IBIDI
CULTURE DISH WAS USED DUE TO HIGH HEAT TRANSFER CAPACITY. (A-RIGHT) .......................................................15
FIGURE 3 FIRST SET OF 6-WELL PLATES FOR CONFLUENCE DETERMINATION. THE INTENSITY OF COOMASSIE BLUE
STAIN INDICATES CONFLUENCE LEVEL OF CELLS....................................................................................................................17
FIGURE 4: SECOND SET OF 6-WELL PLATES TO SCRUTINIZE CONFLUENCE LEVELS BETWEEN 20.000 AND 30.000
CELLS. THE INTENSITY OF COOMASSIE BLUE STAIN INDICATES CONFLUENCE LEVELS OF CELLS.
PARTICULARLY, HIGH CONFLUENT CELL PARTS ARE APPARENT ON LAST 3 WELLS (27.500)..............................18
FIGURE 5: THE SCATTER PLOT OF THE CONFLUENCE LEVELS 10, 20, 22.5, 25, 27.5, 30 AND 40 X 103
. THE GRAPH
WAS PLOTTED ACCORDING TO AVERAGE VALUES OF EACH CONFLUENCE LEVELS. (APPENDIX B)......................19
FIGURE 6: FIRST SET OF VIABILITY ASSESSMENT OF CRYO-VIAL SAMPLES BY LIVE/DEAD STAINING. THE IMAGE
SHOWS, THREE DIFFERENT LEVELS OF CONFLUENCE (LOW, HIGH, PREFERRED) AND (-) CONTROL. CALCEIN
AM (RED-DEAD), ETHIDIUM HOMODIMER (GREEN-LIVE). THE SCALE BAR...................................................................19
FIGURE 7: SECOND SET OF VIABILITY ASSESSMENT WITH TREHALOSE SUPPLEMENTED CPA CONCENTRATIONS BY
LIVE/DEAD STAINING. IN ADDITION, (+) CONTROL (LEFT INCUBATOR DURING FREEZING PROTOCOL AND (-)
CONTROL ARE DISPLAYED. CALCEIN AM (RED-DEAD), ETHIDIUM HOMODIMER (GREEN-LIVE). THE SCALE BAR
IS 50 𝝁𝒎 (10X) ..................................................................................................................................................................................21
FIGURE 8: THE BOX PLOT OF VALIDATION OF IN-HOUSE CPA. THE LEFT SIDE OF THE BOX PLOT; HIGH, LOW,
PREFERRED (20X103
) DISPLAYS VIABILITY AT THESE CONFLUENCE LEVELS. T-S: TREHALOSE
SUPPLEMENTED BBFM: BAMBANKER FREEZING MEDIA. RIGHT SIDE OF THE BOX PLOT DISPLAYS CELL
VIABILITY OF SUPPLEMENTED SAMPLES AND BBFM AT 20X103
SEEDING DENSITY. CELL VIABILITY IS AS
PERCENTAGE (%)..............................................................................................................................................................................22
FIGURE 9: SET OF VIABILITY ASSESSMENT FOR IN-HOUSE DEVICE EXPERIMENT (AT -80O
C PRE-COOLED COPPER
BLOCK). IN ADDITION 2X (+) CONTROL (LEFT IN INCUBATOR DURING FREEZING) FROM DIFFERENT SITE OF THE
CULTURE DISH AND 2X (-) CONTROL; KILLED BY MEOH AND FROZEN W/O CPA. CALCEIN AM (RED-DEAD),
ETHIDIUM HOMODIMER (GREEN-LIVE). THE SCALE BAR IS 50 𝝁𝒎 (10X).....................................................................26
FIGURE 10: ILLUSTRATION OF VITRIFICATION BY IN-HOUSE DEVICE AND BY OPTIMIZED IN-HOUSE DEVICE. THE
IMPROVEMENT (VITRIFIED AREAS) IS CLEAR.............................................................................................................................27
FIGURE 11: SET OF VIABILITY ASSESSMENT FOR IN-HOUSE DEVICE EXPERIMENT (AT -197O
C PRE-COOLED COPPER
BLOCK) THE IMAGE ILLUSTRATES AFTER VITRIFICATION CELL WERE MAJORLY DEAD. CALCEIN AM (RED-
DEAD), ETHIDIUM HOMODIMER (GREEN-LIVE). THE SCALE BAR IS 50 𝝁𝒎 (10X)......................................................28
FIGURE 12: STRUCTURAL ANALYSIS OF SURFACE DEPENDENT VITRIFICATION SAMPLES BY IMMUNOSTAINING.
NUCLEI (BLUE-DAPI), MICROTUBULES (GREEN-BIOTINYLATED ANTI-MOUSE), ACTIN FILAMENTS(RED/ROSA-
PHALLOIDIN). LEFT HAND PANEL IMAGES WERE TAKEN BY 10X AND RIGHT HAND PANEL SAME IMAGES BY 20X.
THE SCALE BAR IS 50 𝝁𝒎 (10X&20X)......................................................................................................................................29
FIGURE 13: ILLUSTRATION OF IMAGE J WORKFLOW. A-) RAW IMAGE, B-) PROCESSED IMAGE W/O WATERSHED C-)
PROCESSED IMAGE WITH 33% CONFLUENCE (AREA FRACTION)......................................................................................40
List of Table
TABLE 1 TABLE OF THREE MAJOR METHODS OF CRYOPRESERVATION. THE LOW-CPA VITRIFICATION COMBINES ALL
THE ADVANTAGES OF TWO MAJOR APPROACHES WHILE AVOIDING THEIR SHORTCOMINGS. .....................................8
TABLE 2: TABLE OF EXPERIMENTAL SETUP OF IN-HOUSE DEVICE EXPERIMENT, IN WHICH AT -80O
C PRE-COOLED
COPPER BLOCK WAS USED. FOR TREHALOSE CONCENTRATIONS; (+ ) WAS , (-): WAS NOT SUPPLEMENTED. 24
TABLE 3: TABLE OF EXPERIMENTAL SETUP OF IN-HOUSE DEVICE EXPERIMENT, IN WHICH AT -197O
C PRE-COOLED
BLOCK WAS USED. FOR TREHALOSE CONCENTRATIONS; (+ ) WAS, (-) WAS NOT SUPPLEMENTED.. ..................27
TABLE 4: DATA TABLE OF CONFLUENCE DETERMINATION. CONFLUENCE LEVELS WERE DISPLAYED AS PERCENTAGE.
..................................................................................................................................................................................................................41
TABLE 5: DATA SET FOR VALIDATION OF IN-HOUSE CPA. BBFM: BAMBANKER FREEZING MEDIA. T-S: TREHALOSE
SUPPLEMENTED. CELL VIABILITY AS PERCENTAGE................................................................................................................42
4. 4
Table of Contents
ABSTRACT.........................................................................................................................................2
ABBREVIATIONS.............................................................................................................................2
TABLE OF FIGURES ......................................................................................................................3
LIST OF TABLE ................................................................................................................................3
TABLE OF CONTENTS..................................................................................................................4
INTRODUCTION ...............................................................................................................................6
MATERIALS AND METHODS....................................................................................................11
HTERT CULTURE.......................................................................................................................................... 11
CONFLUENCE DETERMINATION.................................................................................................................. 12
STAINING PROTOCOLS.................................................................................................................................. 12
Coomassie Blue..............................................................................................................................................12
Live/Dead.........................................................................................................................................................13
Immunostaining............................................................................................................................................13
VALIDATION OF IN-HOUSE CPA ................................................................................................................. 14
IN-HOUSE DEVICE.......................................................................................................................................... 14
The design........................................................................................................................................................14
The experiment..............................................................................................................................................16
Thermodynamic Analysis..........................................................................................................................17
RESULTS ..........................................................................................................................................17
CONFLUENCE DETERMINATION .................................................................................................................. 17
VALIDATION OF IN-HOUSE CPA.................................................................................................................. 19
SURFACE DEPENDENT VITRIFICATION....................................................................................................... 22
THERMODYNAMIC ANALYSIS ...................................................................................................................... 22
DETERMINATION OF REQUIRED CPA VOLUME ........................................................................................ 23
IN-HOUSE DEVICE EXPERIMENT.................................................................................................................. 24
At -80oC pre-cooled copper block..........................................................................................................24
At -197oC pre-cooled copper block.......................................................................................................27
DISCUSSION....................................................................................................................................30
CONFLUENCE DETERMINATION.................................................................................................................. 30
VALIDATION OF IN-HOUSE CPA ................................................................................................................. 30
SURFACE DEPENDENT VITRIFICATION....................................................................................................... 31
ACKNOWLEDGMENTS ...............................................................................................................33
REFERENCES.................................................................................................................................34
APPENDIX A....................................................................................................................................39
THE ADAPTED WORKFLOW OF IMAGE J..................................................................................................... 39
APPENDIX B....................................................................................................................................41
TABLE OF CONFLUENCE DETERMINATION............................................................................................... 41
TABLE OF VALIDATION OF IN-HOUSE CPA............................................................................................... 42
6. 6
Introduction
Dorsal root ganglion (DRG) neurons (also known as sensory afferent neurons) form
part of the peripheral nervous system transporting signals from the sensory organs to
the appropriate integration centre. While the body of these neurons are located within
discrete ganglia either side of the spinal cord, the axons extend outward toward the
skin, muscles, tendons, joints and internal organs, monitoring touch, stretch and
temperature sensations as well as pain. Hence, DRG neurons are indispensable for
neuroscience and pharmacology research (Melli and Höke, 2009). Currently, the
Centre of Cell Engineering group is working on a way to supply human DRG neurons
for pharmacological testing in pain research. These cells are extremely valuable and
rare to obtain, as they are isolated from deceased donors and, despite working to
improve cell yields, cell viability post-dissociation and during culture remains limited.
As such, storage and transportation of these cells is crucial for their use in research.
For this reason, the basis of my research focused on developing and optimising a
cryopreservation protocol (initially using adherent cell lines) to facilitate the storage
and the transportation of viable, ready-to-use and functionally active DRG neurons.
Cryopreservation is a process of preserving living cells or tissues by cooling them to
sub-zero temperatures, at which the frozen material is genetically stable (viable) and
metabolically inert. One of the problems associated with freezing cells, however, is the
formation of ice crystal during the freezing process, which has a negative impact on
cell viability post-thaw. Crystallisation occurs in a random manner at the freezing point
of the substance and the process consists of two major phases: nucleation and crystal
growth. Nucleation is a stochastic process resulting in the localised assembly of
molecules into clusters in a periodic manner forming the crystal structure. This process
can occur spontaneously but it can be also induced either by vibration or a nucleating
agent (Seggio et al., 2008). Heterogeneous (stochastic) nucleation occurs during a
slow cooling rate around 1o
C/min, however at high cooling rates (-50 to -100 o
C/min)
homogenous nucleation occurs. Ice crystals formed during homogeneous nucleation
are much smaller compared to heterogeneous nucleation (Das, 1992). The crystalline
stage is the lowest energy configuration, however it ensures transition of the molecules
from disordered to a highly ordered state (solid). Following, second phase, the crystal-
growth stage causes size increase based on ice clusters (Bartell, 1995). By minimizing
ice crystal formation through cryopreservation methods, damage to cells can be
avoided and rapid return to normophysiological function of the cells after thawing can
be ensured (Baust et al., 2009).
7. 7
Successful cryopreservation allows long-term storage of cell lines in an inventory e.g.
for neural tissue engineering applications (Higgins et al., 2011; Otto et al., 2003) and
improves the logistics of a variety of medical studies by enabling transport of specific
cell lines (Higgins et. al., 2011; Otto et al., 2003; Karlsson et al., 1996). Moreover,
cryopreservation provides flexibility as well as repeatability for experimental workflow
and accumulates the biomass of healthy, distinct cells for banking as well as for high-
throughput screening (Kaur and Vemuri, 2014). Advanced cooling protocols can be
optimized for each cell type to ensure optimal survival rates (post-thaw). However,
limitations may arise due to the type of cooling strategy e.g. fast or slow cooling,
devices and methodology..
Currently, there are three major cryopreservation procedures described in literature;
slow-freezing, vitrification, ultra-rapid freezing (low-CPA vitrification). Slow freezing is
a step-wise procedure that is carried out over a long time period and uses expensive
instruments (e.g. a CRF-controlled rate freezer). It does not prevent ice crystallization
Figure 1: The illustration of the extended phase diagram of cell preservation at cryogenic
temperature. In addition, the graph also demonstrates the phase diagram of Evaporative drying,
which is used for desiccation of the cells in this study (He, 2011).
8. 8
(homogenous) hence extra- and intracellular ice formation can result in osmotic and
mechanical stress, which is detrimental to cell survival (Figure 1-2) (Pegg, 2005; He,
2011). Vitrification is an ice-free (negligible amount of ice-crystals) freezing process
(Figure 2), in which cryoprotectant agents (CPA) are employed to decrease the glass
transition temperature and hence inhibit ice crystal nucleation as well as protect the
cell against ice-crystals by preventing the actual freezing (amorphous phase) (Fahy et
al., 2009; He, 2011) (Figure 1). Moreover, no appreciable degradation occurs over time
in living matter encompassed within a vitreous matrix and theoretically it could be
applicable to all biological systems. Vitrification works particularly well on small sample
volumes, because small volume increases the cooling rates and reduces level of
required cryoprotectant agent. CPA are usually toxic and may cause osmotic stress
(Table 1) (Beier et al., 2013; Beier et al., 2011). Furthermore, the immersion step
directly into the liquid nitrogen (Ln2) to reach high cooling rates is vitally injurious to the
cells and may cause contamination (Table 1) (Lopez et al., 2012; Higgins et al., 2011;
Szurek and Eroglu, 2011; Best, 2015; Beier et al., 2013; Beier et al., 2011),.
Lastly, ultra-rapid freezing, also called Low-CPA vitrification (Figure 1), creates ultra-
fast cooling rate with low amounts of cryoprotectant agent and without programmable
instruments. The underlying rational is if the cooling rate increases, the less amount of
cryoprotectant is required to achieve vitrification (Figure 1- Table1). Several studies
have demonstrated that this procedure has a low efficiency due to formation of
Table 1 Table of three major methods of cryopreservation. The low-CPA vitrification combines all the
advantages of two major approaches while avoiding their shortcomings.
9. 9
intercellular ice-crystals compared to the other techniques (Kuleshova and Lopata,
2002; Chong et al., 2009; AbdelHafez et al., 2010). However to surpass this deficiency,
various devices (Deutsch et al., 2010; Li et al., 2014; Choi et al., 2015) and procedures
(Chakraborty et al., 2011; Malpique et al., 2010) have been used for low-CPA
vitrification.
In short, the literature has emphasized the superiority of the vitrification procedure (in
terms of post-thaw viability etc) compared to slow-freezing (AbdelHafez et al., 2010;
Beier et al., 2013; Chong et al., 2009; Choi et al., 2015; Kuleshova and Lopata, 2002;
Rezazadeh Valojerdi et al., 2009; Edgar and Gook, 2012)(Table 1). Further works have
shown that adherent cells in a monolayer of 60-80% confluence (Wagner and Welch,
2010; Masters and Palsson, 2009; Kardami et al., 2013; Sillman et al., 2003) are
optimal for successful vitrification due to adequate gaps between cells within the
monolayer and the increased efficiency of heat transfer, compared to cells that are
highly confluent. Conversely, Fahy et al. (2009) have shown that the vitrification
process can be scaled up from small tissue pieces and cell monolayers to complex
organ level such as a rabbit kidney and brain (McIntyre and Fahy, 2015). Particularly,
the rabbit kidney resulted in a remarkable success. On receiving the organ after
thawing, the function of the kidney fully recovered and the rabbit survived.
As previously mentioned, CPAs are indispensable for vitrification and can be classified
into two different types; permeable (pCPA) and non-permeable (non-pCPA). pCPAs
permeate into the cell and work by replacing the intracellular water as the extracellular
salt concentration rises (creating a concentration gradient; Bakhach, 2009) and hence
provides equal distribution of the protective agent across the cell membrane (Baust et
al., 2009) decreasing the glass transition temperature. Non-pCPAs cannot permeate
into cells and therefore contribute to cell survival from the extracellular environment.
Although the efficiency of non-pCPAs on protection of cells is low, they are non-toxic
to cells and they improve post-thaw cell viability (Lee et al., 2014). Complex sugar
molecules are categorized as non-pCPA’s and, during freezing and dehydration, they
may reduce membrane damage and stabilise the plasma membrane by hydrogen
bonding to polar head groups of membrane lipids, which has vital importance under
dehydrated conditions (Lee et al., 2014; Eroglu et al., 2000). Moreover it has also been
suggested that complex sugars decrease the cytotoxic effect of CPAs on cells,
increase glass-forming ability and viscosity during freezing (Chian and Quinn, 2010).
The most commonly used CPAs are dimethyl sulfoxide (DMSO), glycerol, ethylene
glycol (EG) and 1,2-propanediol (PROH). Additionally, the sugars that have been used
10. 10
as preservatives are trehalose, sucrose, and lactose (Lee et al., 2014; Eroglu et al.,
2000). Furthermore novel approaches for vitrification include transfecting cells with a
trehalose transporter and subsequently spin-drying (Chakraborty et al., 2011). This
study suggested a novel way to improve the evaporative drying (spinning) (Figure 1-
Evaporative Drying) by transfecting the cells with a trehalose transporter. Although the
study resulted in moderate mammalian cell viability of 62% after spin-drying, their
approach to using direct Ln2 immersion to cool the cells down caused a considerably
low cell viability compared to other studies (from 62% to 51%) (Higgins et al., 2011;
Negishi et al., 2002). It should be taken into account that the transfection needed to
introduce the transporter, permanently changes the DNA sequence of the cell and
these changes may alter other aspects of the cell’s phenotype and influence other
functions therefore these cells cannot be transplanted for the treatment of
neurodegenerative diseases.
Deutsch et al. (2010) and Li et al. (2014) used specialized glass chips (cryo-chips),
which were designed to maintain an individual cell during the severe conditions of the
freezing-thawing cycle to limit celluar damage. Both studies resulted in considerably
higher viability (>90%). He and Choi et al. (2008; 2015) developed an ultra-fast cooling
technique with quartz micro-capillaries (QMC) whereas Malpique et al. (2009; 2010)
used an alginate polymer to confine neurospheres for cryopreservation. In detail, the
quartz micro-capillaries have a preferable size, which corresponds with the diameter
of the cells to be frozen and has suitable thermodynamic properties (heat transfer) to
provide ultra-fast freezing when immersed into liquid nitrogen (Choi et al., 2015). On
the other hand, alginate hydrogels are similar to the extracellular matrices of cells and
ensures the protection of the cells against mechanical damage during ice crystal
formation as well as protecting against disrupting the cell-to-cell contacts by
immobilising the cells (Lee and Mooney, 2012; Malpique et al., 2010). All three studies
resulted in 90% viability especially after immersion into Ln2. Moreover, they enabled
the concentration of the CPA to be lowered from 8M (used in conventional vitrification
procedures) to 1.5M.
This study aims to design a vitrification protocol to store and transport small numbers
of primary human DRG neurons. These neurons have been characterised as having a
soma diameter between 20-100 µm (Roberts, unpublished work), which is similar to
muscle cells (30-100 µm) but considerably larger than human fibroblasts (10-15 µm)
and the axons of these cells can extend to more than a meter in length (Lodish et al.,
2000; Huang et al., 2008) although much of the axon length is lost during the process
11. 11
of dissociating the dorsal root ganglia into individual neurons. Furthermore, the
individual neurons are surrounded by satellite cells, which are important in providing
regulatory signals to the neurons themselves, thus more than one cell type has to be
considered. In terms of cryopreservation, DRG neurons present a number of
challenges due to their small numbers, large size, negligible mitotic activity and
complex structure compared to other cells, all of which makes them susceptible to
irreversible damage during the freezing process. During initial experiments, Mr.
Thomas Reekie and Dr.Jemma Roberts employed a standard freezing protocol either
immediately after isolation or after the DRG neurons had recovered for 4 days in vitro.
In these works, there was little recovery post-thaw and a large shift in the surviving
population. Thus, unlike commercially available cell lines, which recover well after
freezing using standard CPA procedures, primary human DRG neurons require a
different approach.
This study investigates the validity of a current in-house cryopreservation protocol and
alternative methods of cryopreservation. The overall structure of this study takes the
form of three major parts including: determining optimum confluency, validation of the
in-house CPA and surface dependent vitrification. Due to the limited availability of
human DRG neurons, initial studies were carried out using an immortalised hTERT
fibroblast cell line (Blitterswijk and Boer, 2014) with the intention to move onto rat DRG
neurons and eventually human DRG neurons.
Materials and Methods
All work with cell lines was carried out in a sterile environment in a laminar flow hood.
Cell fixation was conducted in a fume hood
hTERT Culture
Cells were cultured in a supplemented Dulbecco’s Modified Eagle Medium (DMEM)
(70.8% DMEM, 17.7% Medium 199, 8.85% FBS, 0.88% 100mM sodium pyruvate,
1.77% antibiotic mix) in vented 75cm2
cell culture flasks in a 37°C CO2 incubator at
95% humidity. The sub-culturing process was carried out in a step-wise workflow;
washing with HEPES saline, trypsinizing the cells with trypsin/versene solution (0.05
% Trypsin), addition of DMEM (supplemented) and centrifugation in universal tubes at
12. 12
377g for 4 min. Afterwards the supernatant was poured out gently and the cell pellet
resuspended in DMEM (supplemented). The desired volume of cell suspension was
determined by counting with hemocytometer and re-seeded into a T75 flask, a 6-well
plate, or 35mm glass bottomed cell culture dishes (Ibidi). Finally, the samples were left
to culture for 48h in an incubator at 37°C 5% CO2 and 95% humidity.
Confluence Determination
hTERT cell numbers were calculated using a haemocytometer and cell suspensions
containing 10,000; 20,000; 22.500; 25.000; 27.500; 30,000 and 40.000 cells were
seeded into 3 wells of each 6 well plates which already contained 2ml of DMEM per
well. After 48 hours, the wells were observed using a Motic AE31 inverted phase
contrast microscope, and the confluence levels checked visually using Coomassie
staining and Image J, which was optimized according to workflow of Busschots et al.
(2014). The adapted workflow of Image J to calculate the area fraction of cells
according to light microscopy images for confluence determination (Appendix-A:
Figure 13).
Staining Protocols
Three different staining protocols were conducted in this study.
Coomassie Blue
Coomassie Blue staining protocol was used to determine cell confluency. The fixative
was made by 10ml 37% formaldehyde fixative, 90ml 1x PBS and 2g sucrose. The
media was replaced with fixative and cells incubated for 15min at 37°C after which the
fixative was removed and cells washed 3 times with 1x PBS. The, PBS was removed
and 1 ml Coomassie blue stain was added before washing again with PBS to remove
excess stain. Observations were done according to coomassie blue stained 6-well
plates (Figure 4-5)
13. 13
Live/Dead
For viability assessment, cells were stained using a commercially available
Live/Dead® viability kit from Invitrogen containing Calcein AM and Ethidium
Homodimer. Although, the example dilution protocol (Invitrogen, 2005) recommends
an incubation period of 30 minutes in PBS containing 2µM Calcein AM and 4 µM
Ethidium Homodimer (100%), it was determined that 50% of the stain concentrations
(1µM and 2µM respectively) and 10 minutes of incubation in (unsupplemented) DMEM
provided sufficient results compared to incubation in PBS. Hence, Cells from 6-well
plates and culture dishes were stained using only 50% of the recommended
concentration and PBS also was replaced with using DMEM (unsupplemented). For
imaging of stained cells, a Leica DMIRB Inverted Fluorescence Microscope (FITC filter
for Calcein fluorescence, TRITC for Ethidium) was employed. Five pictures were taken
from each culture well at five random positions in order to determine an average
viability. Living and non-living cells were counted by observation and by image J.
Finally, viability was calculated as follows;
% 𝑉𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 =
𝑁𝑜. 𝐿𝑖𝑣𝑒 𝐶𝑒𝑙𝑙𝑠
𝑁𝑜. 𝑇𝑜𝑡𝑎𝑙 𝐶𝑒𝑙𝑙𝑠
𝑥 100
Immunostaining
Cells were fixed with 1x 37% formaldehyde fixative in PBS with 2 g sucrose for 15
minutes. Fixative was removed and permeabilising buffer (10.3g sucrose, 0.292g
NaCl, 0.06g MgCl2 (hexahydrate), 0.476g Hepes in 100ml PBS, pH adjusted to 7.2 and
next 0.5ml Triton added) was added and left at 4o
C (fridge) for 5 minutes. The
permeabilising buffer was removed and PBS/1%BSA was added to culture dishes. The
culture dishes were incubated at (37o
C) for 15 minutes and then the PBS/BSA was
removed. The primary antibody (tubulin; supplemented with phalloidin 1:500 (TRITC),
secondary antibody (biotinylated anti-mouse) and Streptavidin-FITC were diluted in
PBS/BSA, 1:100, 1:50 and 1:50 respectively. After each addition of antibody, culture
dishes were wrapped in tinfoil and incubated at 37°C for 1hr. Next, antibodies were
removed and culture dishes were washed 3x with PBS/0.5%Tween. After the
secondary antibody, streptavidin-FITC was added and incubated for 30 min at 4o
C.
The washing step was repeated and a small drop of Vectashield-DAPI was added.
Samples were then stored at 4o
C in PBS. For imaging, a Leica DMIRB Inverted
Fluorescence Microscope was employed
14. 14
Validation of In-house CPA
The desired (preferred: 20x103
), low (10x103
) and high (30x103
) number of cells were
inoculated into 6-well plates and left for 48h cultivation. Wells were washed with 1ml
hepes saline followed by cell detachment with 1ml trypsin (0.05%)/versene. After
detachment, 1ml supplemented DMEM was added to deactivate the trypsin and the
contents of three wells were combined in a universal flask and centrifuged at 377g for
4 minutes. Samples were re-suspended in 600µl of either In-House CPA (20% DMEM,
10% DMSO, 70% FBS), or In-House solutions supplemented with 0.1, 0.5, or 1.0 M
trehalose dehydrate. The concentration of DMSO corresponds low-CPA according to
literature (1.3M). Moreover, cells were also re-suspended in 400µl of either Bambanker
serum free type cell-freezing medium (Lymphotec, Tokyo, Japan). In each set of Cryo-
Vial experiments, one negative and one positive control was prepared. As a negative
control, cells were re-suspended in DMEM (supplemented) prior to freezing while the
positive control cells were left in incubation at 37°C. All samples to be frozen were
transferred to 1.5ml cryo-vials and placed in a CoolCell® LX freezing container at -
80°C for 24 hours. Cells were thawed by incubation in a 37°C water bath for 2-3
minutes. Samples were then inoculated drop-wise into 4ml DMEM (supplemented)
containing universals and flasks were centrifuged for 4 minutes at 377g. Afterwards,
supernatants were poured out and sample pellets were resuspended in 3ml DMEM.
1ml of this suspension was then seeded into one well of a 6-well plate (already holding
2ml DMEM (supplemented)) and left for 48 hours for re-adhesion prior to Live/Dead
staining. Half of the wells of aforementioned positive control was used also for another
negative control, where the cells were deliberately killed by adding 70% MeOH
solution.
In-house device
In order to assess the effectiveness of the in-house design in low-CPA vitrification
further studies were performed using the heat transfer through copper block.
The design
The in-house design was based on the law of thermodynamics (heat transfer
convection – conduction). To form a maximum adiabatic environment (less heat loss),
closed cell polyethylene foam blown with nitrogen (Zotefoam Plastazote® LD33
superior closed cell foam) was used as an insulation material. A 62g gram copper disc
15. 15
was glued to the a cylindrical piece of the in-house device and a 35 mm borosilicate
glass bottomed cell plate was placed upside down to enable direct heat transfer
between the glass bottom and copper disc (Figure 2-A). Copper was used due to its
significantly fast cooling rate (Bald, 1983) and it is readily available.
Figure 2 The illustration of the in-house device design (A). The workflow diagram of the in-house
device (B). The illustration of optimized in-house device to achieve high cooling rates and it is
workflow. For the cultivation of cells the central circle (borosilicate glass) of 35 mm (ibidi) culture
dish was used due to high heat transfer capacity. (A-Right)
ID: 33mm
OD: 35.5mm
Glass Diameter Φ: 23.5mm
height (inside): 7.8mm
Height (outside): 9mm
sssss
ss
C
B
A
16. 16
The experiment
hTERT cells were seeded into the central circle (borosilicate glass) (Figure 2-A) of the
35 mm, high glass bottom µ-dish (ibidi) 20.000 cells (in a similar fashion to well plate
seeding for previous experiments) and left for 48h at 37°C CO2 incubator and 95%
humidity to achieve desired confluence. The media was replaced with either In-house
CPA (20% DMEM, 10% DMSO, 70% FBS), Bambanker, in-house CPA supplemented
with 0.1 and 0.25 trehalose or Bambanker with 0.1M trehalose. The volume of the
media was determined by thermodynamic analysis. Culture dishes were locked (using
the device’s lid mechanism) and inserted into the foam housing of a prototype device.
Cells were frozen by pressing a pre-cooled copper disc (-80°C or -197°C) against the
borosilicate glass bottom of each plate before storing the frozen samples in a -80°C
freezer for 24 hours (Figure 2-B). The copper discs were either pressed against dishes
for 1 minute prior to storage, or for the duration of the storage period.
For samples frozen using a copper block pre-cooled at -80°C, in-house CPA, in-house
CPA supplemented with 0.1M, 0.25M trehalose, Bambanker, Bambanker
supplemented with 0.1M trehalose and DMEM (supplemented, as a negative control)
were used. For –200o
C (liquid nitrogen Ln2) pre-cooled copper block experiment, In-
house CPA, in-house CPA supplemented with 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 1M,
Bambanker, Bambanker supplemented 0.1M, 0.2M, 0.3M, 0.5M, and DMEM (as a
negative control) were used. Due to stiff heat transfer condition between pre-cooled
copper disc and borosilicate glass, the device was optimized through small variations
(Figure 2-C). The cells were thawed by adding pre-warmed culture medium (37o
C)
immediately after separating the copper block from the culture dish. In addition to this,
the temperature change of the Ln2 pre-cooled copper block was estimated by
electronic thermometer (300o
C to -50o
C). Estimation was done through recording time
between -197o
C to -50o
C
17. 17
Thermodynamic Analysis
According to the thermodynamic calculations in the results section, the required
volume of CPA for experiments carried out with pre-cooled copper blocks at -80o
C and
-197o
C was mcpa= 30 𝜇𝑙 and mcpa=40 𝜇𝑙, respectively.
Results
Confluence determination
In order to determine optimal confluence level after 48h cultivation period, coomassie
blue staining and image J analysis were conducted. Together, the results suggested
that the wells seeded with 20.000 cells resulted in preferable confluence level while
Image J analysis indicated that the confluence level of wells seeded at 20,000 cells
was between 55-75% (area fraction) at the end of the experiment, which corresponds
with recommended confluence levels described in a number of cryopreservation
studies (Wagner and Welch, 2010; Masters and Palsson, 2009; Kardami et al., 2013;
Sillman et al., 2003) (Figure 3). Cell densities of 10, 30 and 40 x 103
cells per well were
10.000 hTERT Fibroblast cells
20.000hTERT Fibroblast cells
40.000hTERT Fibroblast cells
30.000hTERT Fibroblast cells
Figure 3 First set of 6-well plates for confluence determination. The intensity of
coomassie blue stain indicates confluence level of cells
18. 18
also processed by Image J (Appendix-A: Appendix-B: Table: 4). After 2 days of
incubation, these wells were considered to be low confluence (35-55%), high (75-90%)
and very high (90-100%) respectively (Figure 3-5). The low confluence level resulted
in large gaps between the cells whereas high confluence level resulted in a multi-layer
formation of the cells instead of a monolayer. To further investigate this, seeding
densities of 20, 22.5, 25 and 27.5 x 103
cells were stained with Coomassie blue and
the cell confluence levels measured by Image J (Appendix-A: Appendix-B: Table 4).
The observations and measurements showed that there was no significant difference
in confluence levels between wells seeded with 20, 22.5 and 25 x 103
cells (Figure 4-
5) while wells seeded with 27.5 x 103
cells demonstrated high confluence (>75%;
Figure 4-5). Consequently, further studies were seeded with 20 x103
hTERT fibroblast
cells.
20.000hTERT Fibroblast cells
22.500hTERT Fibroblast cells
25.000hTERT Fibroblast cells
27.500hTERT Fibroblast cells
Figure 4: Second set of 6-well plates to scrutinize confluence levels between 20.000
and 30.000 cells. The intensity of Coomassie blue stain indicates confluence levels
of cells. Particularly, high confluent cell parts are apparent on last 3 wells (27.500).
19. 19
Validation of in-house CPA
In order to validate the preservative effect of the tested CPAs on cell viability, initial
studies were carried out using a standard, freezing procedure with cryovials. The
(-) Control 70% MeOH
0% cell viability
High confluence (>75%);
70% cell viability
Preferred Confluence (55-75%);
80% cell viability
Low Confluence (35-55%);
60% cell viability
0
20
40
60
80
100
120
-3000 2000 7000 12000 17000 22000 27000 32000 37000 42000
ConfluenceLevel(Area
Fraction(%))
Cell Amount
Confluence Level
Figure 5: The scatter plot of the confluence levels 10, 20, 22.5, 25, 27.5, 30 and 40 x 103
. The graph
was plotted according to average values of each confluence levels. (Appendix B)
Figure 6: First set of viability assessment of Cryo-vial samples by Live/dead staining. The
image shows, three different levels of confluence (low, high, preferred) and (-) control. Calcein
AM (red-dead), Ethidium Homodimer (green-live). The scale bar
20. 20
results of the in-house CPA were compared with commercially available Bambanker
freezing media. In these experiments, the cryovials were frozen at –80 o
C for 24h.
Subsequently, cells were thawed and cultivated 48 hours in 6-well plates. Afterwards,
cell viability was assessed using staining and Image J. Additional experiments were
conducted by supplementing the in-house CPA with 0.1M, 0.5M, 1M trehalose to
investigate the effect of this sugar (as a non-permeable CPA) on cell survival. As per
the first set of experiments, cells were seeded into 6-well plates at densities of 10, 20
and 40 x103
. The 10 x103
and 40 x103
densities were used to observe the negative
effect of high and low confluences on cell viability during freezing.
For samples using CPA not containing trehalose, wells seeded with 20 x103
cells
showed significantly better results with 80% cell viability compared with cultures that
were seeded with 10 x103
and 40 x103
cells, which demonstrated 60% and 70% cell
viability respectively (Figure 6-8, Appendix-B: Table 5). Samples that were frozen in
CPA supplemented with 0.1M, 0.5M and 1M trehalose or Bambanker freezing media,
were similarly assessed. The results indicated that 0.5M and 1M trehalose were too
concentrated for the cells and caused vital osmotic pressure, hence a significantly high
number of burst cells and several detached cell clusters were observed (Figure 7- Top
panel, Figure 9, Appendix-B: Table 5). In comparison, CPA supplemented with 0.1M
trehalose or Bambanker, demonstrated more than 85% and 87% viability respectively
(Figure 7-8, Appendix-B: Table 5). The, negative (-ve) control (cells that were frozen
in DMEM only) showed no living cells after thawing, which was probably as a result of
ice crystal formation (Figurea7-8).
21. 21
1 M Trehalose supplemented in-house CPA
33% cell viability
0.5 M Trehalose supplemented in-house CPA
57% cell viability
0.1 M Trehalose supplemented in-house CPA
85% cell viability
Bambanker Cell Freezing Medium
87% cell viability
(+) Control (left in at 37o
C incubator)
100% cell viability
(-) Control 70% MeOH
0% cell viability
Figure 7: Second set of viability assessment with trehalose supplemented CPA
concentrations by Live/Dead staining. In addition, (+) Control (left incubator during freezing
protocol and (-) Control are displayed. Calcein AM (red-dead), Ethidium Homodimer (green-
live). The scale bar is 50 𝝁𝒎 (10x)
22. 22
.
Figure 8: The box plot of validation of in-house CPA. The left side of the box plot; High, Low,
Preferred (20x103
) displays viability at these confluence levels. T-S: Trehalose supplemented
BBFM: Bambanker freezing media. Right side of the box plot displays cell viability of
supplemented samples and BBFM at 20x103
seeding density. Cell viability is as percentage (%).
Surface dependent vitrification
For vitrification experiments, an in-house device was designed (Figure 2). The design
is consistent with the study of Bier et al (2012), which made use of surface based
cryopreservation in a commercially available µ-dish (Ibidi). ). The design is based on
enhancement of heat transfer by borosilicate glass (Figure 2-A, B) and copper block
(Bald, 1983) to achieve low-CPA vitrification. This approach enables the cells to be
frozen while maintaining cell-cell contact, and minimizing mechanical and chemical
stress by avoiding enzymatic degradation or detachment of colonies. The design is
based on the law of thermodynamics (heat transfer convection–conduction). In order
to form an adiabatic environment (less heat loss), a closed cell polyethylene foam
blown with nitrogen (Zotefoam Plastazote® LD33 superior closed cell foam) was used
as an insulation material.
Thermodynamic Analysis
𝑘copper = 385 𝑊/𝑚𝐾
𝑘borosilicate glass = 1.2 𝑊/𝑚𝐾
𝑘polystrene = 0.033 𝑊/𝑚𝐾
ℎairgap = 40
𝑊
𝑚2
𝐾
(𝑓𝑜𝑟𝑐𝑒𝑑 𝑐𝑜𝑛𝑣𝑒𝑐𝑡𝑖𝑜𝑛 𝑎𝑝𝑝𝑟𝑜𝑥. )
ℎzotefoam = 1.157 𝑊/𝑚2
𝐾 (𝑐𝑎𝑙𝑐𝑢𝑙𝑎𝑡𝑒𝑑)
𝐿copper = 0.01 𝑚
𝐿borosilicate = 0.17 𝑥 10-3
𝑚 (Figure 2-A)
Tin = 25℃ (𝑟𝑜𝑜𝑚 𝑡𝑒𝑚𝑝)
Tout= -80℃ 𝑎𝑛𝑑 − 200℃
-20.00
0.00
20.00
40.00
60.00
80.00
100.00
120.00CellViability(%)
In-house CPA
Validation of In-house CPA
23. 23
Surface area of convection is same for both materials. Hence convection from
Zotefoam block to upper surface of copper block is as follows;
𝑞 = ℎzotefoam (𝑇in − 𝑇1) (1)
Conduction through pre-cooled copper block;
𝑞 = 𝑘copper / 𝐿copper (𝑇1 – 𝑇2) (2)
Convection from the bottom surface of the copper block to the air gap;
𝑞 = ℎairgap (𝑇2 − 𝑇airgap) (3)
Convection from the air gap to surface of the borosilicate glass (bottom of 35 mm
ibidi Culture dish);
𝑞 = ℎairgap (𝑇airgap − 𝑇3) (4)
Conduction through borosilicate glass to material of culture dish (polystyrene)
𝑞 = 𝑘borosilicate / 𝐿borosilicate (𝑇3 − 𝑇4)(5)
Convection from the borosilicate glass to polystyrene;
𝑞 = ℎpolystyrene (𝑇4 − 𝑇out) (6)
According to temperatures; Tin , T1 , T2, T3, Tairgap, Tout, , the equations were
rearranged;
=
(Tin – Tout )
(
1
ℎzotefoam
) + (
𝐿copper
𝑘copper
) + (
1
ℎairgap
) + (
1
ℎairgap
) + (
Lboro
kboro
) + (
1
ℎpolystyrene
)
(7)
Determination of required CPA volume
After determining heat flux by thermodynamic analysis, the required volume of CPA to
cover the inner borosilicate coverslip of the 35 mm (ibidi) culture dish was calculated
through first law of thermodynamics by including energy loss during phase change
(latent heat). Due to a lack of data according to heat of fusion of biological mixtures
(70% FBS), in-house CPA was approximated as water. To find the required CPA
volume with phase change estimations were done between;
First law of thermodynamics,
24. 24
𝑞 = 𝑚𝐶∆𝑇 (8)
and Enthalpy change,
𝑞 = 𝑚. 𝛥𝐻𝑓 (9)
𝛥𝐻𝑓= 334 J/g (heat of fusionwater)
𝐶CPA ≈ 4
𝐽
𝑔o
𝐶
(20% 𝐷𝑀𝐸𝑀, 10% 𝐷𝑀𝑆𝑂, 70% 𝐹𝐵𝑆)
The estimation was done according to a calculated volume without any energy loss
during phase change. The calculated volume interpolated until both equations gave
similar results. As a result, the desired CPA volume was calculated to be:
For a copper block pre-cooled at -80o
C;
𝑚 = 30 𝜇𝑙
For a copper block pre-cooled at -197 o
C;
𝑚 = 40 𝜇𝑙
In-house device experiment
At -80o
C pre-cooled copper block
Firstly, in-house CPA without supplement and Bambanker freezing media were used
for
freezing and storage at -80o
C for 24h. After thawing, only cell debris and a few cell
clusters were seen and, after washing steps for Live/Dead staining, none of the
attached cells were left in the culture dishes. In-house CPA supplemented with 0.1M
trehalose (Table 2) showed several attached cell clusters after washing steps with
>60% cell viability (Figure 9- Top panel). Two images at top panel of Figure 9 were
taken from two different attached cell clusters. Hence, it is understood that trehalose
(none-pCPA) demonstrated a greater protective influence than the in-house CPA
alone. That said, Bambanker supplemented with 0.1M trehalose did not demonstrate
Table 2: Table of experimental setup of in-house device experiment, in which at -80o
C pre-cooled
copper block was used. For trehalose concentrations; (+ ) was , (-): was not supplemented.
At -80 °C pre-cooled copper block Trehalose Supplement
w/o Trehalose 0.1 M 0.25 M
In-house CPA + + +
Bambanker Freezing Media + + -
25. 25
the same result. Following trehalose supplemented in-house design samples (0.25M)
did not demonstrate any similar cell clusters. Although mostly cells were detached (cell
viability decrease (>60%) some cells were left attached and viable (Figure 9 - 2nd
Panel
left).
26. 26
0.1 M Trehalose supplemented
in-house CPA
0.1 M Trehalose supplemented
in-house CPA
(-) Control killed by 70% (MeOH)
(+) Control (left in at 37o
C incubator) Cell
cluster
0.25 M Trehalose supplemented
in-house CPA
(+) Control (left in at 37o
C incubator)
Outer site of cell clusters
0.1 M Trehalose supplemented
Bambanker freezing media
(-) Control freezing w/o CPA
Figure 9: Set of viability assessment for in-house device experiment (at -80o
C pre-cooled
copper block). In addition 2x (+) control (left in incubator during freezing) from different site
of the culture dish and 2x (-) control; killed by MeOH and frozen w/o CPA. Calcein AM (red-
dead), Ethidium Homodimer (green-live). The scale bar is 50 𝝁𝒎 (10x).
27. 27
At -197o
C pre-cooled copper block
Copper blocks were pre-cooled at -197o
C (Ln2 –liquid) and were brought in touch with
culture dishes, in which in-house CPA or Bambanker had been used. Despite the use
of CPAs, freezing (ice-crystal formation) was visible to the naked eye (Figure 10). Next,
cells were frozen using the In-house CPA and Bambanker freezing media
supplemented with trehalose concentration 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 1M and
0.1M, 0.3M, 0.5M, respectively (Table 3). However no living cells were observed.
Using the information from the thermodynamics calculations, I started to optimise the
in-house device by locking the cold temperature to achieve surface based freezing
according to Beier et al. (2013). A 35 mm, glass bottom µ-Dish was placed on top of a
copper block without the lid and an additional Zotefoam insulation foam was added to
keep the temperature inside the device more constant. Hence partially apparent
vitrification was achieved (Figure 10). The survival and retained attachment of some
cell clusters was observed however, after Live/Dead staining, it was understood that
few cells were left alive (Figure 11) in all culture samples with any of the different CPAs
and trehalose concentrations used (Table 3). To test if this was due to the CPAs used
or the method itself, commercially available vitrification solutions (Kitazato) were used
however, vitrification could not be achieved.
Table 3: Table of experimental setup of in-house device experiment, in which at -197o
C pre-
cooled block was used. For trehalose concentrations; (+ ) was, (-) was not supplemented..
At -197 °C pre-cooled
copper block Trehalose Supplement
w/o
Trehalose
0.1
M
0.2
M
0.3
M
0.4
M
0.5
M
1.0
M
In-house CPA + + + + + + +
Bambanker Freezing
Media + + - + - + -
In-house Device Optimized In-house device
Figure 10: Illustration of Vitrification by in-house device and by optimized in-house
device. The improvement (vitrified areas) is clear.
28. 28
.
The thermodynamic effects of pre-cooled copper block on cells were investigated
using immunofluorescence to observe the cytoskeletal structure of the cells post-thaw
(Figure 12). This demonstrated that the cells developed a disrupted microtubule
network and deformed nuclei.
0.5 M Trehalose Supplemented
in-house CPA
0.5 M Trehalose supplemented
Bambanker freezing media
0.5 M Trehalose Supplemented in-house
CPA
0.5 M Trehalose supplemented
Bambanker freezing media
Figure 11: Set of viability assessment for in-house device experiment (at -197o
C pre-cooled
copper block) The image illustrates after vitrification cell were majorly dead. Calcein AM
(red-dead), Ethidium Homodimer (green-live). The scale bar is 50 𝝁𝒎 (10x)
29. 29
0.5 M Trehalose Supplemented in-house
CPA
0.5 M Trehalose supplemented
Bambanker freezingmedia
1 M Trehalose Supplemented in-house CPA
(+) Control (left in at 37o
C incubator)
Figure 12: Structural analysis of surface dependent vitrification samples by
immunostaining. Nuclei (Blue-DAPI), Microtubules (Green-Biotinylated anti-mouse),
Actin filaments (red/rosa-phalloidin). Left hand panel images were taken by 10x and
right hand panel same images by 20x. The scale bar is 50 𝝁𝒎 (10x&20x).
30. 30
Discussion
The present study was conducted to investigate the use of different cryopreservation
reagents and develop a robust cryopreservation method in order to establish a way to
preserve human DRG neurons. Unfortunately the study could not be conducted using
DRG neurons due to the limited timescale of the project; hence all experiments were
conducted with hTERT fibroblast cells. The study was begun from the basis of standard
cryopreservation protocol.
Confluence Determination
First of all, it was determined that a cell seeding density of 20 x103
cells per well in a
6-well plate or 35mm culture dishes resulted in 55-75% cell confluence after 2 days of
incubation.
Validation of In-house CPA
Cells were frozen at -80o
C to check the effect of the in-house CPA on cell viability. The
results were measured against a commercially available CPA from Bambanker.
Beforehand, the effect of low and high cell confluence on cell viability post-thawing
were also investigated. As expected, a seeding density of 20.000 cells (with 55-75%
confluence) resulted in a higher cell viability compared to seeding densities of lower
and higher densities. This is consistent with a number of published studies (Wagner
and Welch, 2010; Masters and Palsson, 2009; Kardami et al., 2013; Sillman et al.,
2003). To try and improve cell viability, the in-house CPA was supplemented with the
non-pCPA (trehalose), which resulted in a slight increase in viability (80-85%).
According to the results, it can be inferred that in-house CPA and in-house CPA
supplemented with 0.1M trehalose is sufficient for the cryopreservation and storage of
hTERT cells at 55-75% confluence using a standard cryovial-based approach at -80o
C.
The increase in cell viability observed in CPA supplemented with 0.1M trehalose could
be attributed to the protective influence of the sugar observed at low temperatures (<-
20o
C). Hence, Trehalose was maintained membrane integrity and was improved cell
viability (Eroglu et al., 2000; Lee et al., 2014). On the other hand,, it was seen here
that high sugar concentration (0.5M and 1M) affected cell viability adversely possibly
due to osmotic stress. Together, this study validates the direct freezing of hTERT cells
in cryovials at -80o
C using in-house CPA, which corroborates studies carried out by Li
31. 31
(2014); and Day and Stacey (2007) with fibroblasts, Wang et al. (2012) with
mesenchymal stem cells, and Lee et al (2013) with murine sperm stem cells.
Surface dependent vitrification
The immersion of cells into liquid nitrogen causes a rapid change in temperature (T)
that causes permanent injury to delicate cell types (e.g. primary neurons) and also
direct immersion into liquid nitrogen increase the risk of contamination (Lopez et al.,
2012; Higgins et al., 2011; Szurek and Eroglu, 2011;Best, 2015; Beier et al., 2013). In
this study, a copper block was used to allow greater control over temperature
compared to direct immersion into liquid nitrogen. The idea was based on pre-cooling
the copper block to -80°C or -197ºC and bringing it into contact with cells on the glass
bottom of the culture disc. Hence, cooling occurs regarding thermodynamics, either at
high (-197o
C - 25o
C= -225ºC) or at moderate (-80o
C -25o
C = -105°C) cooling rate,
which ensures the stratified heat transfer compared to direct liquid nitrogen immersion.
All in all, the in-house prototype was designed to facilitate the surface dependent
vitrification process (low-CPA vitrification) without direct contact with liquid nitrogen
and it is compatible for storing cells over extended periods in -80°C.
Firstly, the in house device was used with a pre-cooled copper disc at -80°C and at -
197°C to utilize surface vitrification. It is significant that vitrification occurs lower than -
137C (Figure 1) hence at -80°C pre-cooled copper block experiment was conducted
to see the effect of the in-house design on surface dependent freezing. Surprisingly,
the first set of experiment samples (-100°C:ΔT) with Bambanker or in-house CPA
showed no cell viability. Attached cells came off after washing steps for Live & Dead
staining. A possible explanation for the detachment might be hypothermic shock due
to the high difference in temperature between the cells and the copper (McCance and
Huether, 2015). The high rate of heat transfer due to the capacity of copper at the pre-
cooled stage might be a factor contributing to the hypothermic shock (Bald, 1983). To
try and improve cell viability, the in-house CPA was supplemented with trehalose.
Some of the samples demonstrated several attached clusters with >60% cell viability
(Figure 9, Top panel) so a study was carried out with different trehalose concentrations
in both freezing media (in-house CPA and Bambanker). Although the viability of cells
was lower compared positive controls (left 48h for cultivation; Figure 8), it is most likely
that the trehalose concentration increases cell viability. A possible explanation for this
could be that the cryopreservative effect of trehalose has a substantial role in surface-
dependent freezing protocol. Following the experiments with the in-house design using
pre-cooled copper blocks at -196ºC was to validate the design of surface-dependent
32. 32
vitrification. Samples that did not contain trehalose in the CPA resulted in poor cells
viability (only cell debris). This could be because of limitations in design, which fail to
keep the low temperature (<-137o
C, Figure 1) or in-house CPA is ineffective at high
cooling rates (<-137o
C). To determine whether the CPA was the problem,
commercially available CPAs from Bambanker and Kitazato were also investigated
however the result was the same. Next, the in-house design was optimized according
to Beier et al. (2013) (Figure 2-C) and the CPAs were supplemented with trehalose.
Although there was significant decrease in viability compared to controls, the results
strengthened the idea that trehalose addition and the modifications (Figure 2-C) made
to the in-house device to improve heat transfer, increases cell viability (Figure 10-11).
According to this result, it is noticed that trehalose reduces membrane damage and
stabilise the cell structure in dehydrated conditions (Eroglu et al., 2000; Lee et al.,
2014).
The cytoskeletal structures of several dead cell clusters in different samples were
examined. Figure 12 shows that the microtubules of the cell cytoskeleton ruptured
possibly due to intracellular ice formation. Moreover, the nuclei of the cells were
wrinkled, granulated and condensed indicative of nuclear damage (Hendriks et al.,
2015). These findings corroborates with the high amount of dead cells and
detachments according to viability assessments (Figure 11). A possible explanation
for these results may be the lack of adequate effect of permeable CPA resulting in
intracellular ice-crystallization as a direct result of the CPA being unable to fully mix
with a cell monolayer compared to a standard cryovial approach using suspended
cells. Another possible explanation for this is that the temperature increased drastically
(10-12o
C in 5 sec) when the copper block was removed from the freezer during the
preparation of in-house device. The advantage of using the copper block is its high
capacity, which provides high-cooling rate when pre-cooled (Bald, 1983), but this may
be a disadvantage when removing the copper from the freezer resulting in a lower
cooling temperature than -137o
C thus resulting in intracellular ice-crystallization.
In conclusion, these findings will doubtless be scrutinised as each step of the
vitrification process is interdependent and thus could not be optimized independently
within the limited time scale of this project. However, the present results are significant
in at least two major respects. Firstly, in-house CPA is sufficient for direct freezing at -
80o
C. Secondly, in-house CPA is most likely practicable for surface dependent freezing
at -80o
C. Hence, this study validates the in-house CPA using a standard Cryovial
freezing approach at -80°C and also using the in-house design for Surface dependent
33. 33
freezing at -80°C On the other hand, the results related to vitrification experiments
(low-CPA vitrification) were not very encouraging. The in-house design could not
achieve fast cooling rate, hence total vitrification could not be observed. A further study
with more focus on the in-house design is therefore suggested. All in all, the
experiments should be repeated to generate reliable data.
Acknowledgments
Firstly, I would like to express my sincere gratitude to my supervisors Dr. Mathis Riehle
and Dr. Jemma Roberts for the continuous support, motivation and guidance on my
Master’s degree research. Without their precious support it would not be possible to
conduct this research. Secondly, I would like also thanks to Dr. Tao Sun from
Loughborough University Chemical Engineering department for his support for
thermodynamic analysis. Lastly, I would like to show gratitude Mrs Carol-Anne Smith,
regarding her help for understanding basic experimental procedures.
34. 34
References
AbdelHafez, F.F., Desai, N., Abou-Setta, A.M., Falcone, T., Goldfarb, J., 2010.
Slow freezing, vitrification and ultra-rapid freezing of human embryos: a
systematic review and meta-analysis. Reproductive BioMedicine Online 20,
209–222. doi:10.1016/j.rbmo.2009.11.013
Bakhach, J., 2009. The cryopreservation of composite tissues. Organogenesis
5, p. 119–126.
Bald, W.B., 1983. Optimizing the cooling block for the quick freeze method. J
Microsc 131, p. 11–23.
Bartell, L.S., 1995. Nucleation Rates in Freezing and Solid-State Transitions.
Molecular Clusters as Model Systems. J. Phys. Chem. 99, p. 1080–1089.
doi:10.1021/j100004a005
Baust, J.G., Gao, D., Baust, J.M., 2009. Cryopreservation. Organogenesis 5,
p. 90–96.
Beier, A.F.J., Schulz, J.C., Dörr, D., Katsen-Globa, A., Sachinidis, A.,
Hescheler, J., Zimmermann, H., 2011. Effective surface-based
cryopreservation of human embryonic stem cells by vitrification. Cryobiology
63, p. 175–185. doi:10.1016/j.cryobiol.2011.06.003
Beier, A.F., Schulz, J.C., Zimmermann, H., 2013. Cryopreservation with a twist
– Towards a sterile, serum-free surface-based vitrification of hESCs.
Cryobiology 66, p. 8–16. doi:10.1016/j.cryobiol.2012.10.001
Best, B.P., 2015. Cryoprotectant Toxicity: Facts, Issues, and Questions.
Rejuvenation Res 18, p. 422–436. doi:10.1089/rej.2014.1656
Blitterswijk, C.V., Boer, J.D., 2014. Tissue Engineering. Academic Press. 17,
p. 25-35
Busschots, S., O’Toole, S., O’Leary, J.J., Stordal, B., 2014. Non-invasive and
non-destructive measurements of confluence in cultured adherent cell lines.
MethodsX 2, p. 8–13. doi:10.1016/j.mex.2014.11.002
Chakraborty, N., Menze, M.A., Malsam, J., Aksan, A., Hand, S.C., Toner, M.,
2011. Cryopreservation of Spin-Dried Mammalian Cells. PLOS ONE 6,
e24916. doi:10.1371/journal.pone.0024916
Chian, R.-C., Quinn, P., 2010. Fertility Cryopreservation. Cambridge University
Press. 7, p. 26-28
Choi, J.K., Huang, H., He, X., 2015. Improved low-CPA vitrification of mouse
oocytes using quartz microcapillary. Cryobiology 70, p. 269–272.
doi:10.1016/j.cryobiol.2015.04.003
35. 35
Chong, Y.-K., Toh, T.-B., Zaiden, N., Poonepalli, A., Leong, S.H., Ong, C.E.L.,
Yu, Y., Tan, P.B., See, S.-J., Ng, W.-H., Ng, I., Hande, M.P., Kon, O.L., Ang,
B.-T., Tang, C., 2009. Cryopreservation of Neurospheres Derived from Human
Glioblastoma Multiforme. Stem Cells 27, p. 29–39.
doi:10.1634/stemcells.2008-0009
Das, D.K., 1992. Pathophysiology of Reperfusion Injury. CRC Press.11, p. 331-
335
Day, J.G., Stacey, G., 2007. Cryopreservation and Freeze-Drying Protocols.
Springer Science & Business Media. P. 262-265
Deutsch, M., Afrimzon, E., Namer, Y., Shafran, Y., Sobolev, M., Zurgil, N.,
Deutsch, A., Howitz, S., Greuner, M., Thaele, M., Zimmermann, H., Meiser, I.,
Ehrhart, F., 2010. The individual-cell-based cryo-chip for the cryopreservation,
manipulation and observation of spatially identifiable cells. I: methodology.
BMC Cell Biol. 11, p. 54. doi:10.1186/1471-2121-11-54
Eroglu, A., Russo, M.J., Bieganski, R., Fowler, A., Cheley, S., Bayley, H.,
Toner, M., 2000. Intracellular trehalose improves the survival of cryopreserved
mammalian cells. Nat Biotech 18, p. 163–167. doi:10.1038/72608
Fahy, G.M., Wowk, B., Pagotan, R., Chang, A., Phan, J., Thomson, B., Phan,
L., 2009. Physical and biological aspects of renal vitrification. Organogenesis
5, p. 167–175.
Groot, A.S.D., Cohen, T., Ardito, M., Moise, L., Martin, B., Berzofsky, J.A.,
2010. 3 - Use of Bioinformatics to Predict MHC Ligands and T-Cell Epitopes:
Application to Epitope-Driven Vaccine Design, in: Kaufmann, D.K. and S.H.E.
(Ed.), Methods in Microbiology, Immunology of Infection. Academic Press, p.
35–66.
He, X., Fowler, A., Menze, M., Hand, S., Toner, M., 2008. Desiccation kinetics
and biothermodynamics of glass forming trehalose solutions in thin films. Ann
Biomed Eng 36, p. 1428–1439. doi:10.1007/s10439-008-9518-8
Hendriks, W.K., Roelen, B. a. J., Colenbrander, B., Stout, T. a. E., 2015. Cellular
damage suffered by equine embryos after exposure to cryoprotectants or
cryopreservation by slow-freezing or vitrification. Equine Vet. J. 47, p. 701–707.
doi:10.1111/evj.12341
Higgins, A.Z., Cullen, D.K., LaPlaca, M.C., Karlsson, J.O.M., 2011. Effects of
freezing profile parameters on the survival of cryopreserved rat embryonic
neural cells. Journal of Neuroscience Methods 201, p. 9–16.
doi:10.1016/j.jneumeth.2011.06.033
36. 36
Huang, J.H., Zager, E.L., Zhang, J., Groff, R.F., Pfister, B.J., Cohen, A.S.,
Grady, M.S., Maloney-Wilensky, E., Smith, D.H., 2008. Harvested human
neurons engineered as live nervous tissue constructs: implications for
transplantation. J Neurosurg 108, p. 343–347.
doi:10.3171/JNS/2008/108/2/0343
Invitrogen (2005) “LIVE/DEAD® Viability/Cytotoxicity Kit *for mammalian
cells*” Product Information p.2
Kardami, E., Hryshko, L., Mesaeli, N., 2013. Cardiac Cell Biology. Springer
Science & Business Media. 6, p. 113-115
Karlsson JOM, Eroglu A, Toth TL, Cravalho EG, Toner M., 1996.Fertilization
and development of mouse oocytes cryopreserved using a theoretically
optimized protocol. Hum Reprod;11:p. 1296–305.
Kaur, N., Vemuri, M.C., 2014. Neural Stem Cell Assays. John Wiley & Sons. 8,
p. 67.
Kuleshova, L.L., Lopata, A., 2002. Vitrification can be more favorable than slow
cooling. Fertil. Steril. 78, p. 449–454.
Lee, K.Y., Mooney, D.J., 2012. Alginate: properties and biomedical
applications. Prog Polym Sci 37, p. 106–126.
doi:10.1016/j.progpolymsci.2011.06.003
Lee, Y.-A., Kim, Y.-H., Ha, S.-J., Kim, B.-J., Kim, K.-J., Jung, M.-S., Kim, B.-G.,
Ryu, B.-Y., 2014. Effect of sugar molecules on the cryopreservation of mouse
spermatogonial stem cells. Fertil. Steril. 101, p. 1165–1175.e5.
doi:10.1016/j.fertnstert.2013.12.033
Lee, Y.-A., Kim, Y.-H., Kim, B.-J., Kim, B.-G., Kim, K.-J., Auh, J.-H., Schmidt,
J.A., Ryu, B.-Y., 2013. Cryopreservation in Trehalose Preserves Functional
Capacity of Murine Spermatogonial Stem Cells. PLOS ONE 8, e54889.
doi:10.1371/journal.pone.0054889
Li, L., Lv, X., Guo, H., Shi, X., Liu, J., 2014. On-chip direct freezing and thawing
of mammalian cells. RSC Adv. 4, p. 34443–34447. doi:10.1039/C4RA06972B
Lodish, H., Berk, A., Zipursky, S.L., Matsudaira, P., Baltimore, D., Darnell, J.,
2000. Molecular Cell Biology, 4th ed. W. H. Freeman, p. 276-290.
Lopez, E., Cipri, K., Naso, V., 2012. Technologies for Cryopreservation:
Overview and Innovation, in: Katkov, I. (Ed.), Current Frontiers in Cryobiology.
InTech. p. 526-535
Malpique, R., Ehrhart, F., Katsen-Globa, A., Zimmermann, H., Alves, P.M.,
2009. Cryopreservation of adherent cells: strategies to improve cell viability and
37. 37
function after thawing. Tissue Eng Part C Methods 15, p. 373–386.
doi:10.1089/ten.tec.2008.0410
Malpique, R., Osório, L.M., Ferreira, D.S., Ehrhart, F., Brito, C., Zimmermann,
H., Alves, P.M., 2010. Alginate encapsulation as a novel strategy for the
cryopreservation of neurospheres. Tissue Eng Part C Methods 16, p. 965–977.
Doi:10.1089/ten.TEC.2009.0660
Masters, J., Palsson, B.Ø., 2009. Human Adult Stem Cells. Springer Science
& Business Media 7. p. 48-55
McCance, K.L., Huether, S.E., 2015. Pathophysiology: The Biologic Basis for
Disease in Adults and Children. Elsevier Health Sciences. p. 76-80
McIntyre, R.L., Fahy, G.M., 2015. Aldehyde-stabilized cryopreservation.
Cryobiology 71, p. 448–458. Doi:10.1016/j.cryobiol.2015.09.003
Melli, G., Höke, A., 2009. Dorsal Root Ganglia Sensory Neuronal Cultures: a
tool for drug discovery for peripheral neuropathies. Expert Opin Drug Discov 4,
p. 1035–1045. doi:10.1517/17460440903266829
Methods in Microbiology, 1971. . Academic Press. 13, p. 50-65.
Negishi, T., Ishii, Y., Kawamura, S., Kuroda, Y., Yoshikawa, Y., 2002.
Cryopreservation of brain tissue for primary culture. Exp. Anim. 51, p. 383–390.
Otto F, Görtz P, Fleischer W, Siebler M. Cryopreserved rat cortical cells
develop functional neuronal networks on microelectrode arrays. J Neurosci
Methods 2003;128:p. 73–81.
Pegg, D.E., 2005. The role of vitrification techniques of cryopreservation in
reproductive medicine. Hum Fertil (Camb) 8, p. 231–239.
doi:10.1080/14647270500054803
Rezazadeh Valojerdi, M., Eftekhari-Yazdi, P., Karimian, L., Hassani, F.,
Movaghar, B., 2009. Vitrification versus slow freezing gives excellent survival,
post warming embryo morphology and pregnancy outcomes for human cleaved
embryos. J Assist Reprod Genet 26, p. 347–354. doi:10.1007/s10815-009-
9318-6
Seggio, A.M., Ellison, K.S., Hynd, M.R., Shain, W., Thompson, D.M., 2008.
Cryopreservation of transfected primary dorsal root ganglia neurons. Journal
of Neuroscience Methods 173, p. 67–73. doi:10.1016/j.jneumeth.2008.05.017
Sillman, A.L., Quang, D.M., Farboud, B., Fang, K.S., Nuccitelli, R., Isseroff,
R.R., 2003. Human dermal fibroblasts do not exhibit directional migration on
collagen I in direct-current electric fields of physiological strength. Experimental
Dermatology 12, p. 396–402. doi:10.1034/j.1600-0625.2002.120406.x
38. 38
Szurek, E.A., Eroglu, A., 2011. Comparison and Avoidance of Toxicity of
Penetrating Cryoprotectants. PLOS ONE 6, e27604.
doi:10.1371/journal.pone.0027604
Thomson, H., (2016), Ark of the immortals: The future-proof plan to freeze out
death, New Scientists, 3080, 2 July 2016
Wagner, A.K., Boninger, M.L., Levy, C., Chan, L., Gater, D., Kirby, R.L., 2003.
Peer review: issues in physical medicine and rehabilitation. Am J Phys Med
Rehabil 82, p. 790–802. doi:10.1097/01.PHM.0000087607.28091.B7
Wagner, K., Welch, D., 2010. Cryopreserving and Recovering of Human iPS
Cells using Complete KnockOut Serum Replacement Feeder-Free Medium. J
Vis Exp. doi:10.3791/2237
Wang, Y., Bai, J., Chen, J., Liu, L., Wang, Y., 2012. [Comparative studies on
different cryopreservation protocols of human amniotic fluid-derived
mesenchymal stem cells]. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi 26, p.
141–145.
Xiaoming He (2011). Preservation of Embryonic Stem Cells, Methodological
Advances in the Culture, Manipulation and Utilization of Embryonic Stem Cells
for Basic and Practical Applications
Zedgar, D.H., Gook, D.A., 2012. A critical appraisal of cryopreservation (slow
cooling versus vitrification) of human oocytes and embryos. Hum. Reprod.
Update dms016. doi:10.1093/humupd/dms016
39. 39
Appendix A
The adapted workflow of Image J
The optimized Image J analysis steps according to Busschots et al (2014) as follow:
- Open image (file: open) : unprocessed image
- Check the image type (image type 8-Bit)
- Subtract the image background (depends on the quality of the image(process subtract
background)
o Rolling ball radius (20-30): decided interval for hTERT fibroblast cells
o Light background
- Sharpen (process sharpen): in order to disambiguate the cell borders
- Thresholding (imageadjustthreshold)
o Bottom bar should be less than exponential increase part of histogram
o Top bar 90-100%
- Watershed (processbinarywatershed) : in order to separate overlaps
- Background noise (processnoise remove outliers
o Radius (20-30)
o Threshold (50-70)
o Outliers (bright)
- Set measurement (Analyseset measurements)
o Marked boxes; Areas, Standard deviation, integrated density, fraction (area), perimeter,
median, min&max, gray value
o None of the bottom boxes is necessary
- Confluence measurement (Analyse analyse particles)
o Marked boxes; Display results, summarize, include holes
o Size (100-infinity) ,
o Circularity ( 0-1 or 0-2) : depends of the formation of cells
o Show (nothing :w/o watersheds, outlines: with watershed)
- Results (Summary Area fraction : the confluence level of the cells in the image
- The image unprocessed image (imagecolorchange color (any)) : Accuracy check
- Type change (imagetypeRGB)
- Now it is possible to merge images to check the accuracy by image calculator
o Image1: original
o Image2: processed image
- For each culture dish (ibidi, 35 mm) or each well of 6-well plates, 5 images from random points
were taken. From these images, the average confluence levels were calculated.
40. 40
Figure 13: Illustration of Image J workflow. A-) Raw image, B-) processed image w/o watershed C-) processed
image with 33% confluence (area fraction)
A
B C C
