Culture expansion of adipose derived stromal cells. A closed automated Quantum Cell Expansion System compared with manual flask-based culture

Abstract

Background: Adipose derived stromal cells (ASCs) are a rich and convenient source of cells for clinical regenerative therapeutic approaches. However, applications of ASCs often require cell expansion to reach the needed dose. In this study, cultivation of ASCs from stromal vascular fraction (SVF) over two passages in the automated and functionally closed Quantum Cell Expansion System (Quantum system) is compared with traditional manual cultivation.

Methods: Stromal vascular fraction was isolated from abdominal fat, suspended in α-MEM supplemented with 10% Fetal Bovine Serum and seeded into either T75 flasks or a Quantum system that had been coated with cryoprecipitate. The cultivation of ASCs from SVF was performed in 3 ways: flask to flask; flask to Quantum system; and Quantum system to Quantum system. In all cases, quality controls were conducted for sterility, mycoplasmas, and endotoxins, in addition to the assessment of cell counts, viability, immunophenotype, and differentiation potential.

Results: The viability of ASCs passage 0 (P0) and P1 was above 96%, regardless of cultivation in flasks or Quantum system. Expression of surface markers and differentiation potential was consistent with ISCT/IFATS standards for the ASC phenotype. Sterility, mycoplasma, and endotoxin tests were consistently negative. An average of 8.0 × 10⁷ SVF cells loaded into a Quantum system yielded 8.96 × 10⁷ ASCs P0, while 4.5 × 10⁶ SVF cells seeded per T75 flask yielded an average of 2.37 × 10⁶ ASCs-less than the number of SVF cells seeded. ASCs P1 expanded in the Quantum system demonstrated a population doubling (PD) around 2.2 regardless of whether P0 was previously cultured in flasks or Quantum, while ASCs P1 in flasks only reached a PD of 1.0.

Conclusion: Manufacturing of ASCs in a Quantum system enhances ASC expansion rate and yield significantly relative to manual processing in T-flasks, while maintaining the purity and quality essential to safe and robust cell production. Notably, the use of the Quantum system entails significantly reduced working hours and thereby costs.

Keywords: Adipose derived stromal cells; Bioreactor; Cell culture; Cell expansion; Clinical application; Coating; Cryoprecipitate; Mesenchymal stem cell; Storage.

Fig. 1

Flowchart of the experimental setup. Comparability of ASC culture expansion, P0 and P1 in flasks (F) and Quantum Cell Expansion System (Q)

Fig. 2

Flow cytometry immunophenotyping of culture-expanded ASCs P0 and P1 in flasks and Quantum System (n = 2). The expression is shown as percentage positive cells. Results are expressed as (mean ± SEM)

Fig. 3

Impact of the coating time. Two bioreactors were coated with cryoprecipitate in 4 and 24 h, respectively. a After 3 weeks cultivation, the same amount of cells was harvested from both Quantums with similar viability and PD. b ASC phenotype assessed by flow cytometry on ASCs seeded in Quantum coated for 24 h compared to ASCs seeded in Quantum coated for 4 h. The expression is shown as percentage positive cells

Fig. 4

Storage of ASCs. a Cell viability was measured every hour, while ASCs were stored for 5 h in 3 different suspensions: 1% HA, 20% HA or 20% HuS at either RT or 4 °C. b Visual observation of cells stored in 3 mentioned storage media at RT. c ASCs from all storage conditions were seeded in culture flasks. Morphology and cell attachment ability of ASCs was evaluated after 24 h under a microscope. Representative phase contrast images at ×10 magnification. HA human albumin, HuS human serum, RT room temperature