Cell culture engineering, advanced...2017/11/09 · Bioreactor consideration for animal cell culture •Well-controlled homogenous environmental conditions (temperature, pH, DO, etc.,)

Cell culture engineering, advanced

- Bioreactor for large-scale culture -

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Large scale cultivation systems for animal cell culture (Bioreactor systems)

• To manufacture, or to produce many kinds of biological materials (vaccines, antibodies, etc.,)

Necessities

Background

• Some complex biological molecules produced in recombinant bacteria did not exhibit the desire function or activity, because of lack of post-translational modifications. (erythropoietin, etc.,)

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Bioreactor consideration for animal cell culture

• Well-controlled homogenous environmental conditions (temperature, pH, DO, etc.,)

• A large support material surface - volume ratio (for anchorage-dependent cells)

• Efficient removal of toxic products of metabolism, such as lactic acid and ammonium.

• A high-cell density culture and high unit productivity. • The reactor should be gently aerated and agitated, because mammalian cells are very shear sensitive.

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Type of bioreactor

• Roller bottle • Stirred tank • microcarrier • microcapsule • cell aggregate • Hollow fiber system • etc.

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Roller bottle

• A cylindrical screw-capped bottles mostly made of disposable plastic.

• The bottles revolve slowly (between 5 and 60 revolutions per hour) which bathes the cells that are attached to the inner surface with medium.

• Roller bottles are used for the cultivation of both suspension cells and anchored cells. However, for suspension cells, it is usually more convenient to grow the cells in a stirred vessel.

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Roller bottle

BD FalconTM Roller Bottle

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N. Kobayashi, Journal of hepato-biliary-pancreatic surgery (2009) vol. 16 (2) pp. 113-7

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Roller bottle

• Cultivating large quantities of anchorage-dependent cells using essentially the same culture techniques as with traditional cell culture flasks but with considerably less labor.

• The gentle agitation prevents gradients from forming within the medium that may adversely affect growth.

Advantage:

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Stirred tank

• Stirred tanks, or conventional fermentors, have been widely used for culturing suspension cells since the 1960s.

• An axial impeller is often mounted at the top of the bioreactor to drive the liquid downward in large-scale cell culture bioreactors.

• With the use of microcarriers, conventional or macroporous ones, adherent cells can also be cultured in a stirred tank.

• In mammalian cell culture, the primary purpose of agitation is to keep cells or microcarriers suspended in nutrient medium relatively uniformly.

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Stirred tank

Figure 3. 3-liter Cell Optimizer System (Wheaton) with microcarriers

http://www.fibercellsystems.com/support_technical.htm

CORNING Proculture

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Microcarrier

• The basic concept is to allow cells to attach to the surface of small suspended beads so that conventional stirred tank bioreactors can be used for cell cultivation.

• The use of microcarriers for cell culture was first demonstrated by Van Wezel (1967).

• To ease suspending these cell- laden microcarriers, their diameter and density are usually in the range of 100‒300 μm and 1.02‒1.05 g/cm3 respectively.

• This diameter range also gives a good growth surface area per reactor volume. Even at a moderate microcarrier concentration, in the range of 8‒15% culture volume being occupied microcarriers, a signicantly larger surface area per reactor volume can be achieved than that in roller bottles.

Microcarrier: a supporting matrix allowing anchorage-dependent cells attachment and growth in a suspension bioreactor system.

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MicrocarrierMicrocarrier: a supporting matrix allowing anchorage-dependent cells attachment and growth in a suspension bioreactor system.

http://www.fibercellsystems.com/support_technical.htm11

Microcarrier

Biotechnology and Genetic Engineering Reviews, 6:1, 404-439, DOI: 10.1080/02648725.1988.10647854

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Microporous microcarrier

• Macroporous microcarriers contain large internal pores.

• The void space inside allows cells to be cultivated not only on the external surface but also internally.

• Cells in the interior are less susceptible to mechanical damage caused by agitation and gas sparging.

• Being in the interior of microcarriers, cells are more likely to be subject to oxygen limitation due to the long diffusional distance.

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Microporous microcarrier

• diameter：130-380μm!

• average pore size：20μm

Cultispher (Percell Biolytica)

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Hollow fiberHollow fiber membrane: A hollow-type fiber made of a semi-permeable membrane

※高分子ミクロ写真集「目で見る高分子　1．分子集合のかたちと働き」　高分子学会編

Application: water treatment, blood purification (dialysis), plasma separation, etc.,

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Hollow fiber bioreactor

• A hollow fiber bioreactor consists of a bundle of capillary fibers sealed inside a cylindrical tube.

• A hollow fiber system can be used for anchorage-dependent and suspension cells.

• In general, the medium passes through the lumen of the fibers (tube side). The cells are cultured on the outer surface of hollow fibers, or in the extracapillary space (shell side).

• However, "vice versa" is also possible.

• The hollow fiber system can provide a large amount of surface area in a small volume. The cells can grow on and around the fibers at a high-density.

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Dixit V et al., Scan J Gastroenterol Suppl. 220, 101-114, 1996

http://www.genengnews.com/gen-articles/novel-uses-for-hollow-fiber-bioreactors/3700/17


http://www.fibercellsystems.com/support_technical.htm

Figure 2: Typical automated hollow-fiber perfusion bioreactor (HFPB) in operation; this commercially available system maintains 1011 total cells in a chemically defined and animal-product–free media for many months.

MediumReservoir

MediumReservoir

for SecondCartridge

Pump

Harvest Ports(Extracapillary Space)

Hollow-Fiber Cartridge

GasExchanger

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Specific application of bioreactors

• Mass production for therapeutic and diagnostic products

• Regenerative medicine

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Cell culture engineering, advanced-Bioartificial liver-

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Liver: one of a vital organ

Functions of native liver! Metabolic function (Glucose metabolism, etc.) ! Detoxication (Alcohol metabolism, Drug metabolism, etc.) ! Excretion (Bile secretion) ! etc.

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Medical need for liver assist devices

Liver: an important organ with complex functions

• Orthotopic liver transplantation is the only effective therapy for many patients with liver failure. However, there is a limited supply of donor livers, the recipients are subjected to a life of costly immunosuppressive drugs, and this therapy is very expensive at more than $100,000.

• Artificial liver support systems, such as plasma exchange, hemodialysis and hemofiltration, have been proposed as a temporary liver support that maintains patient conditions until liver transplantation or allows the native liver to regenerate. However, these treatments do not adequately support the patient because the liver performs many complex functions for survival.

• Much attention is focused on a bioartificial liver support system that uses living hepatocytes.

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Bioartificial liver support system

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Key factors for bioartificial liver

• Cell source

• Cell culture configuration

• Bioreactor design

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Key factors for bioartificial liver

• Cell source

• Established cell lines and primary hepatocytes are candidates.

• Cell lines: easy to handle, expandable, poor functions

• Primary cells: ideal, but difficult to obtain

• Cell culture configuration

• Monolayer culture: its functions rapidly decrease within a few days.

• 3D culture: expression of a high-liver-specific functions for a long period.

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Key factors for bioartificial liver• Bioreactor design

• An artificial liver module must be designed compactly to be installed at the bedside of a patient.

• Important factors in the design of an artificial liver module

• a high cell density culture

• sufficient mass transfer of oxygen and nutrients between cell and medium

• protection from cell damage by high shear stress caused by medium flow

• ease to scale-up and operate using a design with a simple and compact geometry.

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Hybrid artificial liver with multiplated hepatocyte monolayer

• Hepatocytes isolated from an adult dog liver were cultured on collagen coated borosilicated glass

J. Uchino et al., A Hybrid Bioartificial Liver Composed of Multiplated Hepatocyte Monolayer, ASAIO Transactions 34, 972-977, 1988

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Hybrid artificial liver with multiplated hepatocyte monolayer

• 20!10!0.04cmのホウ珪酸ガラスプレートにコラーゲンをコートし，培養担体として使用

J. Uchino et al., A Hybrid Bioartificial Liver Composed of Multiplated Hepatocyte Monolayer, ASAIO Transactions 34, 972-977, 1988

• An artificial liver module was constructed by stacking 200 glass plates with hepatocytes, which were stacked in a transparent acryl resin box.

• Hepatocytes isolated from an adult dog liver were cultured on collagen coated borosilicated glass.

• Total amount of hepatocytes: around 80 g

• The device was applied to anhepatic dogs. This device significantly prolonged survival time.

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Hollow fiber-type bioartificial liver with microcarrier-attached hepatocytes

Rozga J. et al., Development of a Hybrid Bioartificial Liver, Annals of Surgery 217, 502-511, 1993

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• Isolated porcine hepatocytes were attached to collagen-coated dextran microcarriers.

• Cells and microcarriers were incubated overnight in culture medium. • Microcarrier-attached porcine hepatocytes were inoculated into the extra-fiber space of hollow fiber modules.

• Each hollow fiber module consisted of a polycarbonate cylinder (29.1 mm ID, 31.2 mm OD) containing 670 hollow fibers.

• Total amount of hepatocytes: around 50 g

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Hepatoassist

http://biomed.brown.edu/Courses/BI108/BI108_2002_Groups/liver/webpage/Circepg.htm

• The system consisted of a plasma separator, a charcoal column and a hollow fiber module inoculated with microcarrier-attached porcine hepatocytes.

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Clinical trial of Hepatoassist

• HepatAssist was tested in 171 liver failure patients in a Phase II/III prospective, randomized, controlled trial in 11 U.S. and 9 European medical centers.

• This trial did not achieve its primary endpoint (30-day survival) in the overall study population.

• However, patients with fulminant and subfulminant hepatic failure treated with the HepatAssist had a statistically significant survival advantage compared to controls receiving standard medical care. HepatAssist also demonstrated a favorable safety profile.

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Bioartificial liver module using sandwich culture L. De Bartolo et al., Biotechnol Prog 16, 102-108, 2000

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Bioartificial liver module with hepatocytes cultured in polyurethane foam (PUF)

PUF / hepatocyte spheroid culture200 µm

Hepatocytespheroid

PUF skeleton

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Polyurethane foam (PUF) as a culture substratum for 3D culture

Appearance of a PUF plate1.0 cm

Microscopic photograph of PUF

200µm

Skeleton

Thin film

Characteristics of PUF

• PUF is synthesized from isocyanate and polyol. It has a sponge-like macroporous structure with pores comprising smooth thin film (average pore size about 300μm, porosity 98.8%).

• PUF can achieve sufficient mass transfer and high cell density culture because the pores are partially opened and connected with each other.

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Spheroid formation in the pores of PUF

Primary rat hepatocytes Primary dog hepatocytes Primary porcine hepatocytes

200 µm

Primary human hepatocytes

Human embryonickidney cell (293)

Hep G2(Hepatocyte cell line)

200 µm

200 µm200 µm200 µm200 µm

Primary hepatocytes and various animalcells inoculated in PUF pores spontaneouslyform spheroids with the range of 100 to 150µm in diameter. The hepatocyte spheroidscan maintain several important liverfunctions for at least 2 weeks. Thesespheroids can be used as an immobilizedbiocatalyst because they are immobilized atthe bottom surface of PUF pores.

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Design of bioartificial liver module using PUF

capillary (flow channel)

spheroids

• This module consists of a cylindrical PUF block with many capillaries as flow channel for plasma.

• Almost uniformly inoculated hepatocytes spontaneously form the spheroids in PUF macroporous structure between each capillary within about 24 hours after inoculation.

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Extracorporeal circulation system with PUF module

60 ml / min

80 ml / min

20 ml / min

Pf

Pd

Ps 40 ml / min

P

646 ml / min

Animal side( Blood circulation loop )

Hepatocyte culture module side( Plasma circulation loop )

PolygraphTransfusion

Femoralvein

Warm ischemic liverfailure pig ( 25kg )

Plasma

Blood

Superiorvena cava

: Pump

DO, pHelectrode

Gasexchanger

Plasmaseparator

DO electrode

Sampling

Sampling

Module 1L x 2 cylindersPorcine hepatocytes :200g ( 2.0 x 1010 cells )

Pa, Pv, Pf : Pressure gauge

646 ml / min

P

P

P

P

P P

Only 0.9% NaCl solution

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Preclinical trial of PUF-type bioartificial liver

• As preclinical experiments, two modules with 200 g porcine hepatocytes were applied to a pig (25 kg) with warm ischemic liver failure. In the control group using the same system without hepatocytes,

• The increase in blood ammonia was suppressed and was maintained at normal level for more than 20 h.

• The curative effects such as the maintenance of blood glucose, stabilization of vital signs, and urinary excretion and extension of survival time also were observed.

• As a result, this HALSS is available not only to support liver functions but also to stabilize general conditions.

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Current situation of bioartificial liver development

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One of the most difficult problems of using hybrid artificial liver clinically is obtaining a source of cells.

Candidate of cell source

Primary pocine hepatocyte

Primary human hepatocyte

Stem cell Pluripotent stem cell (ES cell and iPS cell) etc.

• Zoonosis infection • Hyper acute immunoreaction

• Limitation of cell suppliance

• Differentiation capability into mature hepatocyte • High proliferation capability

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Documents

Cell culture engineering, advanced...2017/11/09 · Bioreactor consideration for animal cell culture •Well-controlled homogenous environmental conditions (temperature, pH, DO, etc.,)