The document discusses scaling up manufacturing processes for cell cultures, highlighting essential physical and chemical requirements for bioreactors and various methods such as suspension and monolayer cultures. It details the advantages and challenges of different bioreactor designs, including stirred suspensions, airlift fermenters, and NASA bioreactors, while showcasing advancements in technologies for tissue engineering applications. Key points include improvements in cell proliferation and extracellular matrix accumulation, as well as the potential of nonwoven manufacturing techniques for tissue scaffolds.

1. Scale up of manufacturing
process
YANAMALA VIJAY RAJ
Mtech in clinical Eng
IIT MADRAS & CMC VELLORE & SCTIMST

2. Factors in Scaling Up:
•
For appropriate scaleup, the physical and chemical requirements of cells have to be satisfied.
Physical parameters:
i. Configuration of the bioreactor.
ii. Supply of power.
iii. Stirring of the medium.
Chemical parameters:
i. Medium and nutrients.
ii. Oxygen.
iii. pH and buffer systems.
iv. Removal of waste products.

3. Scale Up Process:
• Scale up involves the development of culture systems in stages from (small scale)
laboratory to (large scale) industry. The methodology adopted to increase the scale of a
culture depends on the proliferation of cells and is broadly divided into two categories.
1) Scale up in suspension.
2) Scale up in monolayer.

4. Scale Up in Suspension:
• Scale-up in suspension is the preferred method as it is simpler.
• Scale-up of suspension culture primarily involves an increase in the volume of the culture.
Small scale generally means the culture capacity less than 2 litres volume (or sometimes 5
litres).

5. Stirred suspension cultures:
• It is usually necessary to maintain cell strains in stirred suspension cultures, by
agitation (or stirring) of the medium.
• The stirring of the culture medium is achieved by a magnet encased in a glass
pendulum or by a large surface area paddle.
• The stirring is usually done at a speed of 30100 rpm.
For pluripotent stem cell stirred suspension culture

7. Static suspension cultures:
• Some cells can grow in suspension cultures, without stirring or agitation of the
medium, and form monolayer cells

8. Continuous Flow Culture:
• In a continuous flow culture, it is possible to keep the cells at a desired and set
concentration, and maintain.
• This is carried out Continuous flow culture consists of growing the cells at the midlog
phase, removal of a measured volume of cells, and replacement by an equal volume of
medium. The equipment, specially designed for this purpose has the facility for removal
of the cells and addition of medium.
• The continuous flow cultures are useful for monitoring metabolic changes in relation to
cell density. However, these cultures are more susceptible to contamination.

9. Continuous Flow Bioreactors
for Articular Cartilage Tissue Engineering
• Tissue constructs cultivated in the bioreactor with NaHCO3-supplemented media were
characterized with significantly increased ( p <0.05) ECM accumulation
(glycosaminoglycans a 98-fold increase; collagen a 25-fold increase) and a 13-fold increase
in cell proliferation, in comparison with static cultures.
• Additionally, constructs grown in the bioreactor with NaHCO3-supplemented media were
significantly thicker than all other constructs

10. Aasma A. Khan, B.A., and Denver C. Surrao, M.Sc; The Importance of Bicarbonate and Nonbicarbonate Buffer
Systems in Batch and Continuous Flow Bioreactors for Articular Cartilage Tissue Engineering; TISSUE
ENGINEERING: Part C Volume 18, Number 5, 2012 Mary Ann Liebert, Inc. DOI: 10.1089/ten.tec.2011.0137

11. Aasma A. Khan, B.A., and Denver C. Surrao, M.Sc; The Importance of Bicarbonate and Nonbicarbonate Buffer
Systems in Batch and Continuous Flow Bioreactors for Articular Cartilage Tissue Engineering; TISSUE
ENGINEERING: Part C Volume 18, Number 5, 2012 Mary Ann Liebert, Inc. DOI: 10.1089/ten.tec.2011.0137

12. Airlift Fermenter Culture:
• The major limitation of scaleup in suspension culture is inadequate mixing and gas
exchange. For small cultures, stirring of the medium is easy, but the problem is with
large cultures.
• The design of fermenter should be such that maximum movement of liquid is achieved
with minimum shear to damage the cells.
• This fermenter is extensively used in the biotechnology industry for culture capacities up
to 20,000 litres.

13. Airlift Bioreactor Design for
Chondrocyte Culture
• The basic unit of cartilage, that is, chondrocyte, requires sufficient shear, strain, and
hydrodynamic pressure for regular growth as it is nonvascular tissue.
Variation of the cell count (XF (Flask), XA (ALR)), and
Viability (VF (Flask), VA (ALR)) with reference to time.
It can be observed that the average cell viability is higher
for ALR (89%) compared to the shake flask which is less
than 80%.
Also, the level of cell count for ALR is quite higher, about
12–15%, than that for shake flask grown cells.

14. NASA bioreactor:
• NASA constructed a bioreactor to grow the cells at zero gravity by slowly rotating the
chamber.
• The cells remain stationary and form three dimensional aggregates and this enhances the
product formation. In the NASA bioreactor, there is almost no shear force; hence the cells
are not damaged.
• Homotypic or heterotypic 3D multicellular spheroids provide a more natural cellular
differentiation than 2D monolayer cultures and show improved mimicry of the behavior
and function of actual tissues
• When spheroids are cultured in conventional Petri-dishes or bioreactors, the restricted
nutrient and oxygen diffusion into the spheroids results in a hypoxic, necrotic center in
constructs larger than 1 mm in size which limits the functional properties of the construct.

15. ScaleUp in Monolayer:
• The monolayer culture are anchorage dependent.
• Therefore, for the scaleup of monolayer cultures, it is necessary to increase the surface
area of the substrate in proportion to the number of cells and volume of the medium.
• Suspension cultures are preferred as they are simple.
• The advantages and disadvantages of monolayer cultures are listed.

16. • Advantages:
Change of medium and washing of cells easy.
It is easy to perfuse immobilized monolayer cells.
The cell product formation (pharmaceutically
important compounds e.g. interferon, antibodies) is
much higher.
The same set up and apparatus can be repeatedly
used with different media and cells.
Disadvantages:
 Tedious and costly.
 Require more space.
 Growth of cells cannot be monitored
effectively.
 Difficult to measure control parameters
(O pH, CO etc.)
 For scaleup of monolayer cultures, a wide
range of tissue cultures and system have
been developed.

17. Roller Bottle Culture:
• A round bottle or tube is rolled around its axis (by rollers) as the medium along with
the cells runs around inside of the bottle.
• As the cells are adhesive, they attach to inner surface of the bottle and grow forming
a monolayer.
Roller bottle culture has certain advantages.
 The medium is gently and constantly agitated.
 The surface area is high for cell growth.
 Collection of the supernatant medium is easy.
There are limitations in roller culture
 Monitoring of cells is very difficult.
 Investment is rather high.

18. Multi-surface Culture:
• The most commonly used multisurface propagator of monolayer is Slunclon cell
factory (in short Nunc cell factory). It is composed of rectangular petri dishlike units
with huge surface area (1,00025,000 cm ).
• The units are interconnected at two adjacent corners by vertical tubes.
• The medium can flow between the compartments from one end.

19. RNEST KNIGHT, JR; Multisurface Glass Roller Bottle for Growth of Animal Cells in
Culture; APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1977

20. Microcarrier Culture:
• Monolayers can be grown on small spherical carriers or microbeads (80-300 pm diameter) referred to
as microcarriers.
• The microcarriers are made up of any one the following materials (trade names given in brackets).
 Plastic (acrobeads, bioplas)
 Glass (bioglass, ventreglas)
 Gelatin (ventregel, cytodex3)
 Collagen (biospex, biospheres)
 Cellulose (DE52/53)
 DEAE Dextran (cytodex I, dormacell)
• The microbeads provide maximum surface area for monolayer
cultures.
• This actually depends on the size and density of the beads.
• The cells can grow well on the smooth surface at the solid
liquid interface.

21. Scale-up of human embryonic
stem cell production
• Scale-up of human embryonic stem cell production in a stirred micro-carrier
culture system
• Currently, hES cells are mainly cultured in static tissue plates, which offer a
limited surface and require repeated sub-culturing.
• Stirred culture systems, such as the rotary bioreactor and the spinner flasks,
represent a significant improvement in culture techniques compared to static
systems,
• Cell Cultivation of hES cells in a stirred system with commercial dextran-based
microcarriers coated with denatured collagen (Cytodex™ 3), which provide an
increased surface area for cell adhesion

24. • In terms of cells per growth surface, cells in the spinners grew to a maximum
concentration of 0.21 million cells/cm2 which is 30% more than the maximum
concentration obtained in static plates, 0.16 million cells/cm2.
• However, the most significant difference was observed in terms of cells per
volume of medium. In spinner a maximum concentration of 1.5 x 106 cells/mL was
achieved compared to 0.36 x 106 cells/mL in the static plates

25. Physiologic Pulsatile Flow Bioreactor
Conditioning of Vascular Grafts
• Mechanical conditioning represents a potential means to enhance the biochemical and
biomechanical properties of tissue engineered vascular grafts (TEVGs).
• A pulsatile flow bioreactor was developed to allow shear and pulsatile stimulation of
TEVGs
• Physiological 120 mmHg/80 mmHg peak-to-trough pressure waveforms can be produced
at both fetal and adult heart rates.
• Flow rates of 2 mL/sec, representative of flow through small diameter blood vessels, can
be generated, resulting in a mean wall shear stress of ∼ 6 dynes/cm2 within the 3 mm ID
constructs

26. DERMAGRAFT scaleup
• DERMAGRAFT® is a skin substitute used to help in the wound closure of diabetic foot
ulcers. It is made from human cells known as fibroblasts, placed on a dissolvable mesh
material.

27. Transcyte

28. Perfusion systems
• The flow of medium through the scaffold porosity benefits cell differentiation by
enhancing nutrient transport to the scaffold interior and by providing mechanical
stimulation in the form of liquid shear.
 TransCyte is made using a sealed bioreactor designed as a cassette containing a scaffold
(Biobrane) for the seeding and attachment of dermal fibroblasts.
 The bioreactor can accommodate eight scaffolds which are placed side by side, with the
necessary manifold tubing to provide the cells media in a uniform manner.
 By maintaining a fluid flow within the bioreactor, the cultures are able to form a 3D-matrix
representative of a human dermis. At the end of the growth process, the cassettes can be
sealed and the tissues can be frozen for storage.
TransCyte

29. Dermagraft
• A modified perfusion bioreactor is used for the cultivation of Dermagraft.
• The soft-walled bioreactor contains 8 units per bag and 12 bags are connected by
an injection molded header system, allowing for simultaneous seeding and media
exchange for 96 pieces of tissue engineered skin replacement.

30. Perfusion systems
• The perfusion system can be employed in combination with intermittent hydrostatic
pressure for the cultivation of cartilages, in which a cyclic hydrostatic pressure (0–5 MPa)
can be applied at 0.5–0.3 Hz (Watanabe et al. 2005)
• Double-chamber perfusion reactor is developed to co-culture chondrocytes and
osteoblasts simultaneously in a biphasic scaffold so as to form an osteochondral
construct (Chang et al. 2004).
• The perfusion chamber reactor has also been coupled with mechanical stimulation
(dynamic or static compression) for long-term cultivation of tissue engineered cartilage
(Seidel et al. 2004)

31. Perfusion systems/ Dynamic or static
seeding?
• Dynamic seeding is shown to be superior to static seeding in terms of seeding
efficiency and uniformity (Wendt et al. 2003)
• Dual compartment perfusion system: One compartment applies continuous perfusion of a
cell suspension through the scaffold pores in oscillating directions (Wendt et al. 2003) while
another compartment incorporates dynamic depth filtration seeding operation by
perfusing the cell suspension perpendicularly through the fibrous scaffolds (Feng and Teng
2005).
• These methods have proven to enhance the seeding efficiency and uniformity.

32. concentric cylinder bioreactor
• Concentric cylinder bioreactor aimed at providing low shear stress and a large
growth area to increase construct production
• Uniform seeding of porous polymeric scaffolds with chondrocytes is achieved
with efficiency greater than 95% within 24 h, and the cartilage constructs are well
populated with chondrocytes after 4 weeks of cultivation.

33. rotating-shaft bioreactor (RSB) for two-phase
cultivation of tissue-engineered cartilage
 The rotation moves the chondrocyte/scaffold
constructs between gas and liquid phases in an
oscillating fashion, thus leading to efficient
oxygen and nutrient transfer.
 Furthermore, when the constructs are moving in
the liquid phase, the construct movement relative
to the medium enables easier liquid penetration
into the interior, thus enhancing the nutrient
transfer and imparting more fluid-induced shear
to the interior cells.

34. Nozzle-Less Electrospinning Nanofiber
Technology
• Electrospinning methods for creating nanofibers from polymer solutions have been
known for decades.
• The nozzle-less (free liquid surface) technology opened new economically viable
possibilities to produce nanofiber layers in a mass industrial scale,

35. • The nanofiber formation from a liquid polymer jet in a (longitudinal) electric field.
• It has been theoretically that the dominant mechanism is whipping elongation
occurring due to bending instability.
• Secondary splitting of the liquid polymer streams can occur also, but the final thinning
process is elongation.

36. Electro-spinning Scale up?
• However, the number of jets needed to reach economically acceptable productivity is
very high, typically thousands.
• This brings into play many challenging task, generally related to reliability, quality
consistency, and machine maintenance (especially cleaning).
• The nozzle-less electrospinning solves most of these problems due to its mechanical
simplicity, however, the process itself is more complex because of its spontaneous
multi-jet nature.

37. Nozzle-less technology
• A rotating drum is dipped into a bath of liquid polymer. The thin layer of polymer is
carried on the drum surface and exposed to a high voltage electric field.
• If the voltage exceeds the critical value , a number of electrospinning jets are
generated.
• The jets are distributed over the electrode surface with periodicity given by equation.
• This is one of the main advantages of nozzle-less electrospinning: the number and
location of the jets is set up naturally in their optimal positions.
• In the case of multi-needle spinning heads, the jet distribution is made artificially.
• The mismatch between “natural” jet distribution and the real mechanical structure
leads to instabilities in the process, and to the production of nanofiber layers which are
not homogenous.

38. Nozzle-less technology

39. Creating tissues from textiles
• Electro-spun nonwovens have been used extensively for tissue engineering applications
due to their inherent similarities with respect to fibre size and morphology to that of
native extracellular matrix (ECM).
• However, fabrication of large scaffold constructs is time consuming, may require harsh
organic solvents, and often results in mechanical properties inferior to the tissue being
treated.
• In order to translate nonwoven based tissue engineering scaffold strategies to clinical
use, a high throughput, repeatable, scalable, and economic manufacturing process is
needed S A Tuin Et all 2016 suggest that nonwoven industry standard high throughput
manufacturing techniques (meltblowing, spunbond, and carding) can meet this need.

40. • In the melt-blowing process, the filaments are drawn and accelerated toward the
collector screen via hot air knives, keeping the filaments in a molten state, allowing for
fine fibre attenuation.
• Collected fibres are still in a tacky state, allowing self-bonding between fibres.
• Conversely, in the spun-bond process, as the filaments exit the spin-beam they are
rapidly solidified by cool air before being drawn pneumatically.
• Drawing of filaments in the solid state increases molecular orientation and leads to
improved mechanical properties compared to melt-blowing, typically at the cost of
greater fiber diameter.
• In carding process, short fibres of a few inches in length (staple fibres) are separated
and entangled by a series of specialized combed rollers to form an unbonded web

41. S A Tuin B Pourdeyhimi and E G Loboa1, Creating tissues from textiles: scalable nonwoven manufacturing techniques for fabrication
of tissue engineering scaffolds, Biomed. Mater. 11 (2016) 015017, doi:10.1088/1748-6041/11/1/015017

42. a) Electro-spun b) Melt-blown c) Spun- bound d) Carding

44. Meltblown, spunbond, and carded can
replace electrospun scafflods
• Thereby it is demonstrated by S A Tuin Et all 2016 that meltblown, spunbond, and carded
high throughput nonwoven manufacturing methods are suitable for production of tissue
engineering scaffolds for hASC.
• hASC viability, proliferation, adipogenesis, and osteogenesis were similar to electro-spun
gold standard nonwoven tissue engineering scaffolds.
• These results are promising in the effort to move tissue engineering strategies out of the
lab and into commercial production and clinical use as they allow large quantities of
material to be produced quickly, economically, and with a wide range of controlled fabric
properties.