Presented by Andrea Gough, Solentim at CCE XVI in Tampa, FL (May 2018)

1. Process improvement delivered by a
high efficiency, automated single cell
cloning system
Andrea Gough*, Erika Parkinson, Ian Taylor, Solentim Limited, UK.
Andrea.Gough@solentim.com
*First author
Abstract
Single cell cloning (SCC) and associated cloning efficiency
(colony outgrowth) is currently regarded as an important
and discrete step in stable cell line development and cell
engineering. Even with improved targeted gene editing
like CRISPR/Cas9, many hundreds of single cell clones still
need to be created and analyzed.
For many laboratories, flow cytometry (FACS) or limiting
dilution (LD) is the current method of choice for single
cell deposition, however, each method has its drawbacks.
When using FACS, very high seeding efficiency results
can be achieved but often at the expense of cell survival
and growth due to the harsh treatment of the cells during
processing.WhenusingLD,thereverseisthecase;whereby
good cell survival can be achieved but at low seeding
efficiencies. These intrinsic inefficiencies often result in
a combination of the two processes being implemented
and used on a case by case basis; FACS for robust cell
lines and LD for sensitive cell lines. In addition to these
drawbacks, neither of these processes offer independent
confirmation of the single cell deposition.
In this poster, we introduce a new dedicated SCC platform
called Verified In-Situ Plate Seeding (or VIPS™), which
combines gentle single cell deposition with concurrent
in-situ image verification of the single cell in a well. VIPS
uses a proprietary process of SMART™ LD whereby the cell
droplet is imaged immediately on dispensing in the well to
confirm presence or absence of a single cell (see figure 2).
Introduction
Previous technology
developed by Solentim
successfully addressed
the issue of verifying
clonality and colony
outgrowth monitoring
in cell line development.
However, there remains
a bottleneck upstream
of this - the efficient
dispensing of single cells. The VIPS represents a unique
approach to single cell dispensing:
• Simple to use
• Gentle (low pressure) on cells therefore applicable to a
wide range of cell lines
• Uses statistically accepted distribution models
• Less labour intensive
• Improves seeding efficiencies
• Enhanced clonality report and evidence gathering
• Fluorescence option for validation
The VIPS™ system is a small bench top instrument that is
placed within a standard biological safety cabinet (class II)
for sterility (see figure 1) with all tubing and components
that come into contact with cells easily sterilised or
replaced. This avoids reliance on expensive proprietary
consumable supplies.
The system dispenses a single droplet into an empty well
followed by immediate imaging of the droplet. If a cell
was deposited, the well will be filled with media. If a cell
was not detected, the system will deposit another drop
to the side and image the position again (see figure 3).
The system uses a stack of images to assess the presence
of a cell and this will take place a maximum of 16 times
to effectively ‘re-use’ any empty wells and increase the
frequency of single cell wells.
Figure 2: Flow diagram representation of the VIPS workflow.
Solentim Ltd
VIPS and SMART LD are a trademarks of Solentim.
Other brands or product names are trademarks of their respective holders.
© Copyright 2018 Solentim Ltd. All rights reserved.
Results are reported in real time so that the user can see
which wells have been populated, and this achieves the
entire dispensing of a 96 well plate in approximately 10
minutes (dependent upon the cell density being used).
All together the VIPS efficiently improves the process
of clonal cell line development by combining droplet
imaging, single cell verification, outgrowth assessment
and seeding validation using FL capabilities.
Methods and materials
Five cell lines were tested for seeding and cloning
efficiency using the VIPS:
• 2x CHOZN® pools (supplied by Millipore Sigma, St Louis,
USA)
• DLD-1 (adherent colon adenocarcinoma)
• HCT116 (adherent colorectal carcinoma)
• THP-1 (suspension monocyte-like cell line)
Cell lines were grown as per manufacturers instructions.
The CHOZN pools were grown in AC free conditions.
Cells were diluted to 18,000 cells/ml and seeded to 96well
plates using the VIPS. Plates were then imaged by the
CellMetric (brightfield) on days 0, 2, 7 and 10 to assess
outgrowth. HCT116, DLD-1 and THP-1 were also cloned
by limiting dilution for comparison.
Results and discussion
CHOZN pool
seeding and
outgrowth data
Seeding efficiencies for
both pools resulted in an
average of 52% of wells
containing a single cell
(see figure 4).
Clonal outgrowth for
the two pools was an
average of 75% and 62%
(see figure 5) compared
to 30-50% and 60-
70% respectively using their normal limiting dilutions
approach (results not shown).
Figure 6 displays a specific example from a plate seeded
withaCHOZNpool. 85%ofwellswereconfirmedtohavea
single cell at the time of seeding (green wells). Outgrowth
monitoring revealed 65% of these cells divided and
formed colonies with a large range in colony size after 13
days (see figure 7). All data is available to the user and can
be used to track and monitor cell growth from the moment
the cell is dispensed into a well.
SystemValidation
In addition to the brightfield ca-
pabilities to monitor colony out-
growth, the VIPS has fluorescent
imaging capability. Utilising fluo-
rescent cells the VIPS data can be
validated using the QC application
on day 0 using the whole well im-
age. In this case outgrowth is not
necessary for results saving the
user time and money.
Presented at: CCE – XVI Conference, Tampa, May 2018
DISPENSE
Small cell droplet
deposited into
empty well
Under low pressure
Continual cell agitation
96 well plate
DETECT
Image droplet
BF standard imaging
Fluorescence imaging for
qualification
CONFIRM
Image feedback loop
No cell = Repeat cell
droplet
Cell present = Media fill
Figure 1: VIPS System location in
biological safety cabinet (class II).
Figure 3: Three images captured of a well being processed by the VIPS
during a seeding run. A) An empty well before dispensing. B) The same
well with a single drop deposited – highlighted by an arrow in the image.
C) The same well after cell detection; the well is
filled with the media.
2. AFTER
DISPENSING
3. MEDIA
FILLING
1. BEFORE
DISPENSING
Figure 4: Average seeding results
obtained by dispensing two CHOZN
pools using the VIPS system into
7 x 96 well plates per pool.
Pool 1 Pool 2
0
20
40
60
Percentage%
Average Seeding Efficiency
P1 P2 P3 P4 P5 P6 P7
0
10
20
30
40
50
60
70
80
90
Percentage(%)
Cloning Efficiency Average Cloning Efficiency
P1 P2 P3 P4 P5 P6 P7
Cloning efficiency
A) CHOZN pool 1
B) CHOZN pool 2
Figure 5: Outgrowth results obtained by dispensing two CHOZN pools using
the VIPS system into 7 x 96 well plates per pool. The orange line indicates the
average cloning efficiency obtained across the seven plates for each pool.
Figure 7: Left image – well B8. Right image - well D10. Growth Rate graph
displaying the growth rate as determined using whole well confluence %.
The whole well image and the confluence readings highlight the difference
in clonal growth between single seeded cells.
Hour 2 Day 2 Day7 Day 9 Day 13
0.00
5.00
10.00
15.00
20.00
Confluence%
B8 D10
Figure 9:
Well output
verifying the
presence on of
a single cell, a
well containing
multiple cells
and a well
containing no
cells using the
fluorescence
imaging
capabilities
of the VIPS.
Figure 8: Graphical representation of the improvement achieved when
using the VIPS to seed HCT116, DLD1 and THP1 cell lines compared to
standard limiting dilution. Data is shown as number of clonal colonies
produced with VIPS and limiting dilution for each cell line and the
resulting percentage increase using VIPS over limiting dilution.
HCT116 DLD1 THP1
0
5
10
15
20
25
30
35
LD- Average # Clonal Growth VIPS - Average # Clonal Growth
Averagenumberofclonalcolonies
per96wellplate
increase in the production of clonally derived colonies
using VIPS vs limiting dilution for alternative cell lines
Figure 6: Software output
from VIPS seeding results
(left image) and whole well
imaging (right image) of
a 96 well plate at point of
seeding and at 13 days post
seeding to show colony
growth. The images captured
of well D8 are also displayed
at each time point to show
the tracking of a single cell
into a colony and how the
software can report this data.
Figure 10:
Enhanced
clonality report
displaying the
VIPS drop image
containing
evidence of
the single cell
combined with a
whole well image,
taken at day 8,
containing the
growing clone a
single or multiple
whole well images
at selected time
points can be
included in
the report.
Figure 11: A) Cell reservoir. B) Media tubing. Both made from materials
suitable for sterilisation and provided as consumables if required.
HCT116, DLD-1 andTHP-1 cell line data
Cell line development and the requirement to dispense
a single cell is required for many applications. To
demonstrate the versatility and sensitivity of the VIPS,
three additional cell lines were seeded using the VIPS and
outgrowth monitored.
For all the cell lines dispensed we saw a marked increase
in the number of clonal colonies achieved per plate with
VIPS seeding vs standard limiting dilution (see figure 8).
Enhanced Clonality Report
The VIPS system generates an enhanced clonality report
that will include the drop image containing the single cell
andsubsequentwholewellimagesofthecolonyoutgrowth
(see figure 10). This report is time and date stamped and
can be produced as a PDF or PowerPoint.
Consumables
TheVIPSsystemoffersuserstheoptiontoeliminatetheneed
for expensive proprietary consumables (see figure 11), the
only parts that come into contact with the cell culture and
media are the cell reservoir and media tubing. Both can be
sterilised and re-used if required and Solentim can provide
thesepartsasconsumables.AlternativelyCellReservoirsand
tubing can be provide as“Project Kits”and treated as single-
use disposables
The data presented here show that the VIPS improves
the cloning and outgrowth efficiency of cell lines
not just for therapeutics but potentially across many
other applications, when compared to FACS and
limiting dilution. Integrating this system into a cell line
development laboratory will provide increased output of
clonesforassessment,clonalityassuranceandsupporting
documentation.