Continuous Mass Production of Epithelial Cells Using a Bioreactor System for Regenerative Medicine

Lewis Ho, PhD

BioReactor Sciences LLC

Keratinocytes derived from epidermis, oral mucosa and urothelium are used in the construction of cell based tissue engineering and regenerative medicine applications. Several methods (Oliveira and Hodges 2005; Bavister et al. 2005; Mignone et al. 2010; Lei and Andreadis 2008; Hodgkinson et al. 2010) are being developed to obtain cells with functional plasticity to construct artificial tissue for transplantation, to correct specific systemic diseases and as a source for cell-mediated wound healing therapies. But a method to grow adult somatic cells with maximum plasticity, from human tissue, that circumvents many of the well-known and currently debated ethical and scientific problems associated with use of embryonic derived stem cells or induced pluripotent stem cells, has not yet been developed. Traditional monolayer culture techniques utilizing trypsin for harvesting the cells results in small quantities of cells and as the cells from each monolayer are expanded by passage the ability of the daughter cells to divide is diminished (Hayflick phenomenon). Also, traditional monolayer culture techniques have several risks such as low efficiency of operations since the process is highly dependent upon manual labor; contamination of the culture and deleterious drift (genotypic or phenotypic) possibly due to the changing environmental conditions resulting from traditional manual culture techniques.

Marcelo et al 2012 has shown that human epithelial keratinocytes in primary culture can be induced by tissue culture manipulation to produce, without the use of enzymes for passaging, large numbers of small cells in a combined suspension/monolayer culture using traditional culture technique with regular T-flasks as shown in Fig.1. They refer to the small cells as e-PUK (epithelial Pop-Up Keratinocyte) cells. They (Peramo et al 2013) also found that many other strains of cells including neonatal cells, breast cells and abdominal cells have the same pattern of producing the e-PUK cells. This method would significantly improve the production of keratinocyte cells without damage of enzymatic treatment and also enhance production efficiency. The traditional culture technique however only allows the production of e-PUKs from the first passage of keratinocyte monolayer for 7 or less days and requires generating another monolayer from the e-PUK generated from the first passage of cells to continue the subsequent production of e-PUK. The life of the subsequent monolayers and number of passages get shorter and burns out within few passages of monolayer due to the lack of ability to properly optimize the cell growing condition using these traditional techniques.  Additionally these traditional techniques require substantial labor and manual operation in an open system which is subject to greater risk of contamination.

A new culture technique utilizing a flask bioreactor was thus reported by A. Miyazawa et al (2018) using a modified T flasks with a novel programmable rocking device. The result showed that this automated bioreactor system has extended culture longevity and proliferative capacity in normal primary human keratinocytes for over 29 days with only one parent monolayer cells. As the floating ePUK cells attached the surface, the cells returned and retained the original characteristics of the keratinocytes.  The schematic diagram of this process is shown in Fig 2.

The system was further modified and improved as shown in Figure 3.

An integrated automatic T-flask bioreactor using a novel rocker and a modified T-75 flask was further improved to perform the process shown above in FIG3. The system comprises an integrated rocker 8 with digital panel 9 mounted with a single T-flask (such as Corning T75) 10, a gas mixture system 11 to feed the gas mixture through inlet port 12 and exit from port 13 through the dispensing system 14 comprising several pinch valves to external designated containers 19, 20 with outlet air filters inside of a CO2 incubator or a refrigerator 15, a pumping system 16 to pump fluid (medium, seed, cell detaching solutions etc.) from the storage containers 17 through inlet port 12 for a fixed volume of 30 ml after the platform 18/vessels 10 return to the horizontal position, the content of flask 10 is programmed based upon the glucose consumption rate to empty and/or harvest by tilting the platform 18 to an angle and through the dispensing system 14 to direct the harvest line to external designated containers such as T-flask 19 or bottle20 inside of a CO2 incubator or a refrigerator 15, the lid 21 covers the flask 10 and sits on platform 18, the temperature of the enclosure is controlled with a hot air heater 22, a portable image monitoring device 23 such as Lonza’s CytoSmart placed on the platform remotely monitored, recorded and controlled by a PC 24.

The process started from a single monolayer growth of primary epithelial cells isolated from adult human epidermis or oral mucosa or ureters at T-flask 10 with intermittent replacement of standard volume of fresh medium (e.g. 15ml in T75 flask) every two to three days from the fresh medium bottle 17 automatically. The spent medium was discarded or saved for analysis. As the monolayer reached greater than 80% growth, the T-flasks were replaced with 2x volume of fresh medium (e.g. 30 ml in T75 flask) automatically and production of e-PUK cells began.  The gas mixture was regulated to a less oxygen tension (<21% O2, e.g. 5%) through 11 and fed to the system through 12 at constant gas flow rate to the system.  Initially, the monolayer in the T-flasks was cultivated at horizontal position under static condition in the device for two 24 hour cycles and the content of e-PUK cells harvested and replaced with 30 ml of fresh medium in each cycle. The initial and end samples of each cycle were analyzed for glucose concentration. Then the next cycle time was calculated by the following equation and the process proceeded.

The frequency (cycle time) of medium replacement t3 for the next cycle is calculated by the following equation (1):

t3= (C0- Cmin)/(dR + dC2/t2)     

where t1 and t2 are the first and second cycle time of the most recent 2 cycles;

dC2 is the difference of glucose concentration change during the second of the most recent 2 cycles;

C0 is the concentration (mg/dl) of the fresh medium; Cmin is the minimum concentration to be maintained in the culture;

dR= dC2/t2-dC1/t1 is the change of glucose consumption rates between the two previous cycles, cycle 1 & 2, where dC1 is the same as dC2 but for cycle 1.

The process continued in the same manner for substantially extended time (for months). Each cycle of e-PUK cells was harvested for immediate use at bottle 20 or at another T-flask 19 in a CO2 incubator 15.  The cells collected at T-flask 19 were subsequently further attached, cultivated, harvested, cryopreserved using traditional method for later use.

Figure 4 further illustrates a production bioreactor using modified vessel with multi-layer of surface plates, such as Corning’s Hyperflask, Cellstack or Thermo’s Cell Factory, to perform the semi-continuous process as established above using T-75 flask. The multi-plate structures are comprised of multiple layer of surface plates to increase cell growth surface area (> 25280 cm2) compared to the small surface area of 75 cm2 available in the T-75 flask bioreactor shown in FIG. 3. The process begins with a T175 flask bioreactor 2a using the same protocol with optimal control of nutrient 3a and oxygen 4a as shown in FIG.2 and FIG.3 to continuously produce the e-PUK daughter cells 5 from the P-0 monolayer which are directly used to seed the production bioreactor 2b. The e-PUK cells quickly attach to the multi-plates of 2D surface carrier in the bioreactor 2b and continue the growth and production process using the same protocol applying the same optimal control of nutrient 3b and oxygen 4b as that in the seed bioreactor 2a.  During the seeding and growing process the production of e-PUK cells from the bioreactor 2b is also self-seeding to the available open surfaces along with the e-PUK cells from the seed T175 bioreactor 2a until all complete surfaces are fully occupied. Then the full production process begins. The production bioreactor 2b continues to apply the same optimal control of nutrient 3b and oxygen 4b and produce e-PUK cells 5 and keratinocyte cells 6 from the same P-1 monolayer throughout the entire production process for a greatly expanded time.

In order to achieve the performance of a production bioreactor using the modified vessel with multi-layer of surface plates, such as Corning’s Hyperflask, Cellstack or Thermo’s Cell Factory indicated above, the AMTBR (automated multi-tray bioreactor ) based on our patented HRBR technology for adherent cell cultures using 2D multi-tray culture vessel was established and patented (US10590374 B2). See the below Figures 5 and 6 and YouTube video learn more.

The fully automated AMTBR resolves limitations of current 2D flat culture surface technology by applying the active gassing and automatically performing the entire culture functions including filling, seeding, culturing, medium exchange, emptying, infection/transfection, sampling, cell detachment, and harvesting all in one closed system and in one click. Additionally, it enables to employ various control and process strategies similar to a typical bioreactor. The AMTBR possesses increased functionality and control-ability, while also substantially simplifying/decreasing the number of devices and thus greatly reducing the physical footprint and operating cost compared to the commercial automatic Cell Factory system (AMCF). Lastly, it costs only fraction of the complex AMCF system. The system can periodically monitor the glucose concentration, pH, etc. by off-line analysis with samples; or optionally by continuously monitoring and control (of pH and DO) by a non-invasive optical pH/DO monitoring system such as PreSens’ Featured pH Monitoring System for Bioprocess Development.

References

•       US 8835169  Compositions, Methods and Systems for preparation of a stem cell-enriched cell population. Stephen Feinberg (2014)

•       Characterization of a unique technique for culturing primary adult human epithelial progenitor/“stem cells”  Marcelo et al: BMC Dermatology 12:8 (2012)

•       Characterization of cultured epithelial cells using a novel technique not requiring enzymatic digestion for subculturing;  Peramo et al: Cell Tissue Bank 14:423 (2013)

•       Utilisation of a bioreactor for culture and expansion of epithelial cells without trypsin or enzymes; A Miyazawa et al,  The Chinese Journal of Dental Research, vo; 21, number 1, 2018

•       US Patent Number 10590374 B2 Automatic multi-tray and multi-plate bioreactor systems for adherent cultures; Lewis and Timothy Ho (2020)