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. 2023 Jun 30;12(13):4414.
doi: 10.3390/jcm12134414.

Scale-Up of Academic Mesenchymal Stromal Cell Production

Caroline Laroye  1   2 , Mélanie Gauthier  1   2 , Jessica Morello  1 , Naceur Charif  2 , Véronique Latger Cannard  3 , Céline Bonnet  4 , Alain Lozniewski  5 , Andrei Tchirkov  6 , Natalia De Isla  2 , Véronique Decot  1   2 , Loïc Reppel  1   2 , Danièle Bensoussan  1   2
Affiliations

Scale-Up of Academic Mesenchymal Stromal Cell Production

Caroline Laroye et al. J Clin Med. .

Abstract

Background: Many clinical trials have reported the use of mesenchymal stromal cells (MSCs) following the indication of severe SARS-CoV-2 infection. However, in the COVID19 pandemic context, academic laboratories had to adapt a production process to obtain MSCs in a very short time. Production processes, especially freezing/thawing cycles, or culture medium have impacts on MSC properties. We evaluated the impact of an intermediate cryopreservation state during MSC culture to increase production yields.

Methods: Seven Wharton's jelly (WJ)-MSC batches generated from seven different umbilical cords with only one cryopreservation step and 13 WJ-MSC batches produced with intermediate freezing were formed according to good manufacturing practices. The identity (phenotype and clonogenic capacities), safety (karyotype, telomerase activity, sterility, and donor qualification), and functionality (viability, mixed lymphocyte reaction) were analyzed.

Results: No significant differences between MSC production processes were observed, except for the clonogenic capacity, which was decreased, although it always remained above our specifications.

Conclusions: Intermediate cryopreservation allows an increase in the production yield and has little impact on the basic characteristics of MSCs.

Keywords: Wharton’s jelly; mesenchymal stem cells; scale-up.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
MSC manufacturing process. After MSC isolation from Wharton’s jelly by the explant method, cells were expanded until passage 2. At the end of passage 2, after trypsinization, the cells were either seeded in passage 3 (continuous production) or cryopreserved for further amplification (discontinuous production).
Figure 2
Figure 2
Production data from the MSC manufacturing process. (A) Seven continuous and thirteen discontinuous MSC production processes were analyzed at the end of P3 in terms of the cell number, viability, doubling time, and cumulative population doubling. (B) A paired analysis was also performed between continuous (n = 5) and discontinuous (n = 13) MSC production on the same parameters. (P2: passage 2, CP P3: continuous passage 3, DP P3: discontinuous passage 3, ns: not significant). One color symbol corresponds to one umbilical cord.
Figure 3
Figure 3
Quality control data from the MSC manufacturing process. (Aa) Immunophenotype analysis at the end of P3 after continuous (n = 7) and discontinuous (n = 13) MSC production. Data represent the mean ± SD. (Ab) Immunomodulation capacity of MSCs at P3 between continuous (n = 5) and discontinuous (n = 2) MSC production. (Ac) The clonogenic capacity was evaluated by the colony-forming-unit fibroblast (CFU-F) assay at the end of P3 after continuous (n = 7) and discontinuous (n = 13) MSC production. (Ad) A paired analysis was also performed between continuous P3 (n = 5) and discontinuous P3 (n = 13) production starting from the same P2 (* p < 0.05, CP P3 vs. DP P3). (Ae) Safety controls were performed at the end of P3 after continuous (n = 7) and discontinuous (n = 13) MSC production. (Af,Ag) Osteogenic and adipogenic differentiation capacities were evaluated at the end of P3 after continuous (Af) and discontinuous (Ag) MSC production by the revelation of calcium deposits and lipid vesicles, respectively. (Ba) Viability after thawing continuous (n = 16) and discontinuous (n = 42) MSC production. (Bb) The clonogenic capacity evaluated by the CFU-F assay after thawing continuous (n = 4) and discontinuous (n = 19) MSC production. (P2: passage 2, CP P3: continuous passage 3, DP P3: discontinuous passage 3, ns: not significant). One color symbol corresponds to one umbilical cord.
Figure 4
Figure 4
Comparison between MultiPL30i and MultiPL100i. A comparison between the standard 5% MultiPL30i and MultiPL100i at different percentages (2, 3, 4, and 5%) as a media supplement in terms of cell viability (A), cell proliferation (B,C), immunophenotype (D), and clonogenicity (E) from P1 to P3 was performed. At the end of P3, additional testing for stability (karyotype and telomerase activity) (F) and senescence assays (G) were implemented to compare MultiPL30i and MultiPL100i. (G) Percentage of senescent cells. Data represent the mean ± SD (n = 3).
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References

    1. Thompson M., Mei S.H.J., Wolfe D., Champagne J., Fergusson D., Stewart D.J., Sullivan K.J., Doxtator E., Lalu M., English S.W., et al. Cell Therapy with Intravascular Administration of Mesenchymal Stromal Cells Continues to Appear Safe: An Updated Systematic Review and Meta-Analysis. EClinicalMedicine. 2020;19:100249. doi: 10.1016/j.eclinm.2019.100249. - DOI - PMC - PubMed
    1. Laroye C., Lemarié J., Boufenzer A., Labroca P., Cunat L., Alauzet C., Groubatch F., Cailac C., Jolly L., Bensoussan D., et al. Clinical-Grade Mesenchymal Stem Cells Derived from Umbilical Cord Improve Septic Shock in Pigs. Intensive Care Med. Exp. 2018;6:24. doi: 10.1186/s40635-018-0194-1. - DOI - PMC - PubMed
    1. Laroye C., Gibot S., Reppel L., Bensoussan D. Mesenchymal Stromal/Stem Cells: A New Treatment for Sepsis and Septic Shock?: MSCs in the Treatment for Sepsis and Septic Shock. Stem Cells. 2017;35:2331–2339. doi: 10.1002/stem.2695. - DOI - PubMed
    1. Condor J.M., Rodrigues C.E., Sousa Moreira R.d., Canale D., Volpini R.A., Shimizu M.H.M., Camara N.O.S., Noronha I.D.L., Andrade L. Treatment with Human Whartons Jelly-Derived Mesenchymal Stem Cells Attenuates Sepsis-Induced Kidney Injury, Liver Injury, and Endothelial Dysfunction. Stem Cells Transl. Med. 2016;5:1048–1057. doi: 10.5966/sctm.2015-0138. - DOI - PMC - PubMed
    1. Chen J., Hu C., Chen L., Tang L., Zhu Y., Xu X., Chen L., Gao H., Lu X., Yu L., et al. Clinical Study of Mesenchymal Stem Cell Treatment for Acute Respiratory Distress Syndrome Induced by Epidemic Influenza A (H7N9) Infection: A Hint for COVID-19 Treatment. Engineering. 2020;6:1153–1161. doi: 10.1016/j.eng.2020.02.006. - DOI - PMC - PubMed

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