Scale up of manufacturing processYANAMALA VIJAY RAJMtech in clinical EngIIT MADRAS & CMC VELLORE & SCTIMST

Factors in Scaling Up:•

For appropriate scale up, 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.

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.

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).

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 30 100 rpm.

For pluripotent stem cell stirred suspension culture

Static suspension cultures:• Some cells can grow in suspension cultures, without stirring or

agitation of the medium, and form monolayer cells

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.

Continuous Flow Bioreactorsfor 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

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

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

Airlift Fermenter Culture:• The major limitation of scale up 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.

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.

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.

Scale Up in Monolayer:

• The monolayer culture are anchorage dependent. • Therefore, for the scale up 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.

• 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 scale up of monolayer cultures, a

wide range of tissue cultures and system have been developed.

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.

Multi -surface Culture:• The most commonly used multi surface propagator of monolayer is

Slunclon cell factory (in short Nunc cell factory). It is composed of rectangular petri dish like units with huge surface area (1,000 25,000 cm ).

• The units are interconnected at two adjacent corners by vertical tubes. • The medium can flow between the compartments from one end.

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

Micro carrier Culture:• Monolayers can be grown on small spherical carriers or microbeads (80- 300 pm diameter)

referred to as micro carriers. • The micro carriers are made up of any one the following materials (trade names given in

brackets).

Plastic (acrobeads, bioplas) Glass (bioglass, ventreglas) Gelatin (ventregel, cytodex 3) Collagen (biospex, biospheres) Cellulose (DE 52/53) DEAE Dextran (cytodex I, dormacell)

• The micro beads 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.

Scale-up of human embryonicstem 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

• 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

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 ofTEVGs

• 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

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.

Transcyte

Perfusion systems

• The flow of medium through the scaffold porosity benefits cell differentiation by enhancing nutrient transport to the scaffold interior and by providing mechanicalstimulation 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

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.

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 osteochondralconstruct (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)

Perfusion systems/ Dynamic or static seeding?

• Dynamic seeding is shown to be superior to static seeding in terms of seedingefficiency 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.

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.

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 inthe liquid phase, the construct movement relativeto the medium enables easier liquid penetrationinto the interior, thus enhancing the nutrienttransfer and imparting more fluid-induced shearto the interior cells.

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,

• 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.

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.

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.

Nozzle-less technology

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.

• 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

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

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

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.