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The enzymatic disaggregation by trypsin does not alter cell quality and genomic stability of adipose-derived stem cells cultivated for human cell therapy

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Abstract

There is no consensus between the protocols used for the isolation, maintenance and cultivation of Adipose-derived stem cells (ADSCs) for therapeutic purposes. Thus, was evaluated the maintenance method of ADSCs submitted to enzymatic disaggregation by trypsin. Was made (1st until 10th passage) immunophenotyping, cell differentiation assays, comet assay, differential cell death, apoptosis, cell viability and membrane integrity by flow cytometry.The results showded that trypsinization,did not induce genomic instability, also did not alter the tail moment. The cell death assay, showed that only on the 10th passage there was a significant reduction and was cofirmed by flow cytometry that is apoptosis. The viability showded significant reduction only in 10th passage, this was related to the loss of integrity of membrane, proven by flow cytometry. The quantities varied along the passages (11 × 105 to 2 × 105). Qualitatively, it can be observed that as the number of cells decreases, there is also a reduction in the juxtaposition of ADSCs and increased of the cell size, it is started in 6th passage. In view of the results, it is suggested for more safety, that ADSCs cultured until the 5th passage being used in human transplantation procedures.

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References

  • Agência Nacional de Vigilância Sanitária-ANVISA (2017) Guia de Inspeção em Banco de Células e Tecidos - Boas práticas em células e tecidos. p. 1–341, 2017. https://www.gov.br/anvisa/pt-br/centraisdeconteudo/publicacoes/sangue-tecidos-celulas-e-orgaos/manuais-e-guias/guia-de-inspecao-em-banco-de-celulas-e-tecidos.pdf

  • Al-Ghadban S, Bunnell BA (2020) Adipose tissue-derived stem cells: immunomodulatory effects and therapeutic potential. Physiology 35:125–133

    Article  CAS  PubMed  Google Scholar 

  • Azari H, Rahman M, Sharififar S, Reynolds BA (2010) Isolation and expansion of the adult mouse neural stem cells using the neurosphere assay J Visual Exp

  • Bacakova L et al (2018) Stem cells: their source, potency and use in regenerative therapies with focus on adipose-derived stem cells—a review. Biotechnol Adv 36:1111–1126

    Article  PubMed  Google Scholar 

  • Bansal H, Comella K, Leon J, Verma P, Agrawal D, Koka P, Ichim T (2017) Intra-articular injection in the knee of adipose derived stromal cells (stromal vascular fraction) and platelet rich plasma for osteoarthritis. J Transl Med 15:141

    Article  PubMed  PubMed Central  Google Scholar 

  • Bernardo ME, Locatelli F, Fibbe WE (2009) Mesenchymal stromal cells: a novel treatment modality for tissue repair. Ann N Y Acad Sci 1176:101–117

    Article  CAS  PubMed  Google Scholar 

  • Bianchi F et al (2013) A new nonenzymatic method and device to obtain a fat tissue derivative highly enriched in pericyte-like elements by mild mechanical forces from human lipoaspirates. Cell Transplant 22:2063–2077

    Article  PubMed  Google Scholar 

  • Bora P, Majumdar AS (2017) Adipose tissue-derived stromal vascular fraction in regenerative medicine: a brief review on biology and translation. Stem Cell Res Ther 8:145

    Article  PubMed  PubMed Central  Google Scholar 

  • Brown LL (2018) Adipose-derived stromal stem cells. Advanced procedures for pain management. Springer, pp 489–507

    Chapter  Google Scholar 

  • Carrancio S et al (2008) Optimization of mesenchymal stem cell expansion procedures by cell separation and culture conditions modification. Exp Hematol 36:1014–1021

    Article  CAS  PubMed  Google Scholar 

  • Cattaneo G, De Caro A, Napoli F, Chiapale D, Trada P, Camera A (2018) Micro-fragmented adipose tissue injection associated with arthroscopic procedures in patients with symptomatic knee osteoarthritis. BMC Musculoskelet Disord 19:176

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Coughlin RP, Oldweiler A, Mickelson DT, Moorman CT III (2017) Adipose-derived stem cell transplant technique for degenerative joint disease. Arthrosc Tech 6:e1761–e1766

    Article  PubMed  PubMed Central  Google Scholar 

  • de Carvalho SL et al (2017) All-trans retinoic acid induces mitochondria-mediated apoptosis of human adipose-derived stem cells and affects the balance of the adipogenic differentiation. Biomed Pharmacother 96:1267–1274

    Article  Google Scholar 

  • Debnath T, Chelluri LK (2018) Standardization and quality assessment for clinical grade mesenchymal stem cells from human adipose tissue. Hematol Transfusion Cell Ther 41:7–16

    Article  Google Scholar 

  • Debnath T, Chelluri LK (2019) Standardization and quality assessment for clinical grade mesenchymal stem cells from human adipose tissue. Hematol Transfusion Cell Ther 41:7–16

    Article  Google Scholar 

  • Dompe C, Wąsiatycz G, Mozdziak P, Jankowski M, Kempisty B (2019) Current clinical applications of adipose-derived stem cells in humans and animals. Med J Cell Biol 7:105–111

    Article  Google Scholar 

  • Fathi E, Farahzadi R (2016) Isolation, culturing, characterization and aging of adipose tissue-derived mesenchymal stem cells: a brief overview. Braz Arch Biol Technol 59

  • Fletcher A (2007) The cell membrane and receptors. Anaesthesia Intensive Care Med 8:443–446

    Article  Google Scholar 

  • Gibbs N, Diamond R, Sekyere EO, Thomas WD (2015) Management of knee osteoarthritis by combined stromal vascular fraction cell therapy, platelet-rich plasma, and musculoskeletal exercises: a case series. J Pain Res 8:799

    Article  PubMed  PubMed Central  Google Scholar 

  • Hermeto L et al (2015) Evaluation of pH effects on genomic integrity in adipose-derived mesenchymal stem cells using the comet assay. Genet Mol Res 14:339–348

    Article  CAS  PubMed  Google Scholar 

  • Hermeto L et al (2016) Effects of intra-articular injection of mesenchymal stem cells associated with platelet-rich plasma in a rabbit model of osteoarthritis. Genet Mol Res 15:3

    Article  Google Scholar 

  • Hermeto LC et al (2020) The efficacy of topical insulin application on rat model with burn wounds treated with adipose-derived stem cells. Int J Burns Trauma 10:296

    CAS  PubMed  PubMed Central  Google Scholar 

  • Hudetz D et al (2017) The effect of intra-articular injection of autologous microfragmented fat tissue on proteoglycan synthesis in patients with knee osteoarthritis. Genes 8:270

    Article  PubMed Central  Google Scholar 

  • Hwang ES, Yoon G, Kang HT (2009) A comparative analysis of the cell biology of senescence and aging. Cell Mol Life Sci 66:2503–2524

    Article  CAS  PubMed  Google Scholar 

  • Koh Y-G, Choi Y-J, Kwon S-K, Kim Y-S, Yeo J-E (2015) Clinical results and second-look arthroscopic findings after treatment with adipose-derived stem cells for knee osteoarthritis. Knee Surg Sports Traumatol Arthrosc 23:1308–1316

    Article  PubMed  Google Scholar 

  • Kornicka K, Marycz K, Tomaszewski KA, Marędziak M, Śmieszek A (2015) The effect of age on osteogenic and adipogenic differentiation potential of human adipose derived stromal stem cells (hASCs) and the impact of stress factors in the course of the differentiation process. Oxidative Med Cell Longevity 2015

  • Legzdina D, Romanauska A, Nikulshin S, Kozlovska T, Berzins U (2016) Characterization of senescence of culture-expanded human adipose-derived mesenchymal stem cells. Int J Stem Cells 9:124

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Marędziak M, Marycz K, Tomaszewski KA, Kornicka K, Henry BM (2016) The influence of aging on the regenerative potential of human adipose derived mesenchymal stem cells. Stem Cells Int 2016

  • Markarian CF et al (2014) Isolation of adipose-derived stem cells: a comparison among different methods. Biotech Lett 36:693–702

    Article  CAS  Google Scholar 

  • Miyagi A, Chipot C, Rangl M, Scheuring S (2016) High-speed atomic force microscopy shows that annexin V stabilizes membranes on the second timescale. Nat Nanotechnol 11:783–790

    Article  CAS  PubMed  Google Scholar 

  • Oliveira RJ, Matuo R, Da Silva AF, Matiazi HJ, Mantovani MS, Ribeiro LR (2007) Protective effect of β-glucan extracted from Saccharomyces cerevisiae, against DNA damage and cytotoxicity in wild-type (k1) and repair-deficient (xrs5) CHO cells. Toxicol Vitro 21:41–52

    Article  CAS  Google Scholar 

  • Onoi Y et al (2019) Second-look arthroscopic findings of cartilage and meniscus repair after injection of adipose-derived regenerative cells in knee osteoarthrits: report of two cases. Regen Ther 11:212–216

    Article  PubMed  PubMed Central  Google Scholar 

  • Panni AS, Vasso M, Braile A, Toro G, De Cicco A, Viggiano D, Lepore F (2018) Preliminary results of autologous adipose-derived stem cells in early knee osteoarthritis: identification of a subpopulation with greater response. Int Orthop 1–7

  • Pesarini JR et al (2017) Vitamin D: Correlation with biochemical and body composition changes in a southern Brazilian population and induction of cytotoxicity in mesenchymal stem cells derived from human adipose tissue. Biomed Pharmacother 91:861–871

    Article  CAS  PubMed  Google Scholar 

  • Pesarini JR et al (2018) Calcitriol combined with calcium chloride causes apoptosis in undifferentiated adipose tissue-derived human mesenchymal stem cells, but this effect decreases during adipogenic differentiation. Biomed Pharmacother 108:914–924

    Article  CAS  PubMed  Google Scholar 

  • Roato I et al (2019) Concentrated adipose tissue infusion for the treatment of knee osteoarthritis: clinical and histological observations. Int Orthop 43:15–23

    Article  PubMed  Google Scholar 

  • Spath L et al (2010) Explant-derived human dental pulp stem cells enhance differentiation and proliferation potentials. J Cell Mol Med 14:1635–1644

    Article  CAS  PubMed  Google Scholar 

  • Truong NC, Bui KH-T, Van Pham P (2018) Characterization of senescence of human adipose-derived stem cells after long-term expansion

  • Tsai C-C, Chen Y-J, Yew T-L, Chen L-L, Wang J-Y, Chiu C-H, Hung S-C (2011) Hypoxia inhibits senescence and maintains mesenchymal stem cell properties through down-regulation of E2A–p21 by HIF-TWIST blood. J Am Soc Hematol 117:459–469

    CAS  Google Scholar 

  • Urt-Filho A et al (2016) Mesenchymal stem cell therapy promotes the improvement and recovery of renal function in a preclinical model. Genet Mol Biol 39:290–299

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Vasanthan J, Gurusamy N, Rajasingh S, Sigamani V, Kirankumar S, Thomas EL, Rajasingh J (2021) Role of human mesenchymal stem cells in regenerative therapy. Cells 10:54

    Article  CAS  Google Scholar 

  • Vieira M et al (2014) Therapeutic potential of mesenchymal stem cells to treat Achilles tendon injuries. Genet Mol Res 13:10434–10449

    Article  CAS  PubMed  Google Scholar 

  • Wang Y, Huso D, Harrington J, Kellner J, Jeong D, Turney J, McNiece I (2005) Outgrowth of a transformed cell population derived from normal human BM mesenchymal stem cell culture. Cytotherapy 7:509–519

    Article  CAS  PubMed  Google Scholar 

  • Yokota N, Yamakawa M, Shirata T, Kimura T, Kaneshima H (2017) Clinical results following intra-articular injection of adipose-derived stromal vascular fraction cells in patients with osteoarthritis of the knee. Regen Ther 6:108–112

    Article  PubMed  PubMed Central  Google Scholar 

  • Yokota N, Hattori M, Ohtsuru T, Otsuji M, Lyman S, Shimomura K, Nakamura N (2019) Comparative clinical outcomes after intra-articular injection with adipose-derived cultured stem cells or noncultured stromal vascular fraction for the treatment of knee osteoarthritis. Am J Sports Med 47:2577–2583

    Article  PubMed  Google Scholar 

  • Zou ML et al. (2020) Insights into the role of adipose‐derived stem cells: wound healing and clinical regenerative potential. J Cell Physiol

  • Zuk PA et al (2001) Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 7:211–228

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The present work was carried out with support from the Federal University of Mato Grosso do Sul—UFMS/MEC—Brazil. This study received the financial supported from FUNDECT, CNPq and (CAPES) institutions, thanks for this.

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Authors and Affiliations

  1. Stem Cell, Cell Therapy and Toxicological Genetics Research Centre (CeTroGen), “Maria Aparecida Pedrossian” University Hospital, Brazilian Hospital Services Company (EBSERH), Campo Grande, Mato Grosso do Sul, Brazil

    Laynna de Carvalho Schweich-Adami, Luana Bernardi, Adrivanio Baranoski, Thais de Andrade Farias Rodrigues, Andréia Conceição Milan Brochado Antoniolli-Silva & Rodrigo Juliano Oliveira

  2. Graduate Program in Health and Development in the Central-West Region, Faculty of Medicine, Federal University of Mato Grosso do Sul (UFMS), Mato Grosso do Sul, MS, Brazil

    Laynna de Carvalho Schweich-Adami, Luana Bernardi, Adrivanio Baranoski, Andréia Conceição Milan Brochado Antoniolli-Silva & Rodrigo Juliano Oliveira

  3. Graduate Programme in Genetics and Molecular Biology, Department of General Biology, State University of Londrina (UEL), Londrina, Paraná, Brazil

    Rodrigo Juliano Oliveira

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  1. Laynna de Carvalho Schweich-Adami
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Contributions

LCSA an LB had the idea for the article, performed the literature search, assays, data analysis and write the article; AB and ACMBAS participated with contributions to the conception of the article and made some assays and RJO performed the data analysis and revised the article.

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Correspondence to Rodrigo Juliano Oliveira.

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Schweich-Adami, L.C., Bernardi, L., Baranoski, A. et al. The enzymatic disaggregation by trypsin does not alter cell quality and genomic stability of adipose-derived stem cells cultivated for human cell therapy. Cell Tissue Bank 23, 641–652 (2022). https://doi.org/10.1007/s10561-021-09958-0

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