Transition from static culture to stirred tank bioreactor for the allogeneic production of therapeutic discogenic cell spheres

Abstract

Background: Culturing cells as cell spheres results in a tissue-like environment that drives unique cell phenotypes, making it useful for generating cell populations intended for therapeutic use. Unfortunately, common methods that utilize static suspension culture have limited scalability, making commercialization of such cell therapies challenging. Our team is developing an allogeneic cell therapy for the treatment of lumbar disc degeneration comprised of discogenic cells, which are progenitor cells expanded from human nucleus pulposus cells that are grown in a sphere configuration.

Methods: We evaluate sphere production in Erlenmeyer, horizontal axis wheel, stirred tank bioreactor, and rocking bag format. We then explore the use of ramped agitation profiles and computational fluid dynamics to overcome obstacles related to cell settling and the undesired impact of mechanical forces on cell characteristics. Finally, we grow discogenic cells in stirred tank reactors (STRs) and test outcomes in vitro (potency via aggrecan production and identity) and in vivo (rabbit model of disc degeneration).

Results: Computation fluid dynamics were used to model hydrodynamic conditions in STR systems and develop statistically significant correlations to cell attributes including potency (measured by aggrecan production), cell doublings, cell settling, and sphere size. Subsequent model-based optimization and testing resulted in growth of cells with comparable attributes to the original static process, as measured using both in vitro and in vivo models. Maximum shear rate (1/s) was maintained between scales to demonstrate feasibility in a 50 L STR (200-fold scale-up).

Conclusions: Transition of discogenic cell production from static culture to a stirred-tank bioreactor enables cell sphere production in a scalable format. This work shows significant progress towards establishing a large-scale bioprocess methodology for this novel cell therapy that can be used for other, similar cell therapies.

Keywords: Bioprocess; Cell spheres; Cell therapy; Progenitor cells; Scale-up; Stirred tank bioreactor (STR).

Fig. 1

Investigated culture modalities. a Cells grown in a static suspension culture CellSTACK modality with methylcellulose exhibit the desired sphere phenotype. b Cells grown in waterwheel form into sheets rather than spheres. c Cells grown in Erlenmeyer flasks attach to vessel wall rather than in suspension. d Cells grown in Wave Bioreactor Bag form large rafts rather than spheres. e Cells form spheres when grown at low agitation in STR, however large spheres grow too large in size causing issues with oxygen transport and settling out of solution. f A large portion of cells grown at low agitation in STR attach to vessel surfaces impacting growth dynamics. g Cells grown at high agitation speeds in STR show some sphere growth but predominantly form single cells. h Using a ramped agitation profile with low initial RPM and high final RPM we are able to grow spheres in STR and limit cell attachment

Fig. 2

Variation in hydrodynamic conditions. a CFD models show that hydrodynamic conditions scale with different slopes and curvature based on RPM increase in STR, b by running STR at various conditions and then modelling aggrecan results using the various hydrodynamic slopes, we are able to see max shear rate (1/s) has the strongest correlation between our investigated hydrodynamic conditions and aggrecan expression (p = 0.012)

Fig. 3

CFD models to enable sphere growth. a Correlation between hydrodynamic conditions and cell outputs include models of max shear rate and aggrecan (p = 0.012), average eddy turbulence dissipation and cell settling (p = 0.024), max shear rate and doublings (p = 0.018), and dynamic agitation with sphere size (p = 0.033). b Illustration of shifts in hydrodynamic environment as RPM shifts in dynamic culture conditions. By growing cells at various RPM and measuring outputs we can model effect of hydrodynamic conditions on cells. c Illustration of cell volume fraction calculated from average eddy turbulence dissipation at a single sphere size and RPM. Graphic shows simulated location of cells which can predict cell settling (other RPM, sphere sizes, and scales also simulated). d In order to minimize shear forces while also keeping spheres in suspension as they grow in size, we leverage CFD to develop dynamic agitation profiles

Fig. 4

Cell characteristics in static suspension versus STR modality. a Diagram of split stream growth where 5 cell lines were grown in both 0.25 L STR and static suspension modalities. b Comparable sphere growth in static suspension culture and STR modalities when using ramped agitation. c Mean sphere size is slightly smaller in STR. d Aggrecan expression, and e doublings from day 2 to end of culture across static suspension culture and 0.25 L STR modalities are comparable. f Identity of cells as measured by flow cytometry is comparable between 0.25 L STR and static modalities

Fig. 5

In vivo comparison of IDCT grown in static suspension versus STR culture. a The rabbit study design starts with disc injury, 2 weeks later the discs are dosed, 6 weeks after dosing the study is terminated. b Mean percent change in Disc Height Index (DHI) as measured from X-ray from dosing to termination is shown with standard error. After dosing, the disc height increased slightly in the sham group, more in the vehicle groups, and more substantially in the Cell Therapy groups. c Disc Histology shows height restoration and increase in hydration (white areas within center of red disc) for discs injected with Cell Therapy compared to vehicle or sham

Fig. 6.

0.25 L versus 50 L STR results. a Diagram of our “split stream” growth where 1 donor was grown in both small scale and pilot scale reactors b Comparable sphere growth observed in small scale and pilot scale reactors. Sphere size c is comparable at harvest. d Aggrecan expression is below the qualified limit of detection for both conditions. e Cell doublings from day 2 to end of culture across 0.25 L and 50 L STR are comparable. f Identity and purity of cells as measured by Flow cytometry is comparable between 0.25 L STR and 50 L STR