Dataset Information

Successful isolation of viable stem cells from cryopreserved microfragmented human adipose tissue from patients with knee osteoarthritis - a comparative study of isolation by tissue explant culture and enzymatic digestion.

ABSTRACT:

Purpose

To investigate if viable stem cells could be isolated and expanded from cryopreserved microfragmented adipose tissue (AT) harvested from patients with knee osteoarthritis.

Methods

Microfragmented abdominal AT from knee osteoarthritis patients was cryopreserved at -80 °C in cryoprotectant-medium. The samples were thawed for stem cell isolation by tissue explant culture (TEC) and enzymatic digestion (ED), respectively. Viability, population doublings, and doubling time were assessed by trypan blue staining and flow cytometry. Cell type and senescence-associated β-galactosidase activity were analyzed by flow cytometry. Osteogenic and adipogenic differentiation was assessed quantitatively by Alizarin-Red-S and Oil-Red-O staining, respectively.

Results

Microfragmented AT from 7 patients was cryopreserved for a period of 46-150 days (mean (SD) 115.9 days (44.3 days)). Viable stem cells were successfully recovered and expanded from all patients using both isolation methods with no significant difference in viable population doublings or doubling time from passage 1 to 3 (p > 0.05). Low levels of senescence-associated β-galactosidase activity was detected for both methods with no significant difference between TEC and ED (p = 0.17). Stemness was verified by stem cell surface markers and osteogenic and adipogenic differentiation performance. Adventitial stem cells (CD31^-CD34⁺CD45^-CD90⁺CD146^-), pericytes (CD31^-CD34^-CD45^-CD90⁺CD146⁺), transitional pericytes (CD31^-CD34⁺CD45^-CD90⁺CD146⁺), and CD271⁺ stem cells (CD31^-CD45^-CD90⁺CD271⁺) were identified using both methods. More pericytes were present when using TEC (25% (24%)) compared to ED (3% (2%)) at passage 4 (p = 0.04).

Conclusions

Viable stem cells can be isolated and expanded from cryopreserved microfragmented AT using both TEC and ED. TEC provides more clinically relevant pericytes than ED.

SUBMITTER: Bagge J

PROVIDER: S-EPMC10036689 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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Successful isolation of viable stem cells from cryopreserved microfragmented human adipose tissue from patients with knee osteoarthritis - a comparative study of isolation by tissue explant culture and enzymatic digestion.

Bagge Jasmin J Hölmich Per P Hammer Freja Aabæk FA Nehlin Jan O JO Vomstein Kilian K Blønd Lars L Hölmich Lisbet Rosenkrantz LR Barfod Kristoffer Weisskirchner KW

Journal of experimental orthopaedics 20230323 1

<h4>Purpose</h4>To investigate if viable stem cells could be isolated and expanded from cryopreserved microfragmented adipose tissue (AT) harvested from patients with knee osteoarthritis.<h4>Methods</h4>Microfragmented abdominal AT from knee osteoarthritis patients was cryopreserved at -80 °C in cryoprotectant-medium. The samples were thawed for stem cell isolation by tissue explant culture (TEC) and enzymatic digestion (ED), respectively. Viability, population doublings, and doubling time were ...[more]

PMID: 36952141

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Project description:BACKGROUND: The treatment of tendon lesions with multipotent mesenchymal stromal cells (MSCs) is widely used in equine medicine. Cell sources of MSCs include bone marrow, as well as solid tissues such as adipose tissue. MSCs can be isolated from these solid tissues either by enzymatic digestion or by explant technique. However, the different preparation techniques may potentially influence the properties of the isolated MSCs. Therefore, the aim of this study was to investigate and compare the effects of these two different methods used to isolate MSCs from solid tissues.Equine adipose tissue, tendon and umbilical cord matrix served as solid tissue sources of MSCs with different stiffness and density. Subsequent to tissue harvest, MSCs were isolated either by enzymatic digestion with collagenase or by explant technique. Cell yield, growth, differentiation potential and tendon marker expression were analysed. RESULTS: At first passage, the MSC yield was significantly higher in enzymatically digested tissue samples than in explanted tissue samples, despite a shorter period of time in primary culture. Further analysis of cell proliferation, migration and differentiation revealed no significant differences between MSCs isolated by enzymatic digestion and MSCs isolated by explant technique. Interestingly, analysis of gene expression of tendon markers revealed a significantly higher expression level of scleraxis in MSCs isolated by enzymatic digestion. CONCLUSIONS: Both isolation techniques are feasible methods for successful isolation of MSCs from solid tissues, with no major effects on cellular proliferation, migration or differentiation characteristics. However, higher MSC yields were achieved in a shorter period of time by collagenase digestion, which is advantageous for the therapeutic use of MSCs. Moreover, based on the higher level of expression of scleraxis in MSCs isolated by enzymatic digestion, these cells might be a better choice when attempting tendon regeneration.

Project description:ObjectiveOvarian tissue cryopreservation is one of the crucial options for fertility preservation. Transplantation of cryopreserved ovarian tissue was proven to restore ovarian endocrine function in patients with premature ovarian insufficiency. Ovaries from deceased donors potentially serve as an excellent and readily available tissue for the translational and basic research. In this study, we used ovaries obtained from 5 deceased donors aged 18-26 years, to evaluate the number and quality of ovarian follicles isolated before and after cryopreservation.DesignPreclinical.SettingAcademic biomedical research laboratory.PatientsDe-identified deceased human donors.InterventionsSlow-freeze cryopreservation and thawing.Main outcome measuresFollicle count, follicle density, follicle viability using immunohistochemical staining (TUNEL).ResultsThe follicle density negatively correlated with age in both cryopreserved/thawed and fresh group. A total of 2803 follicles from fresh and 1608 follicles from cryopreserved tissues were classified and analyzed using Hematoxylin and eosin staining. There was no significant difference in the percent of morphologically normal follicles between two groups. TUNEL assay indicated no higher DNA damage in the follicles and the stroma cells after cryopreservation. Morphologically normal preantral follicles were enzymatically isolated from both fresh and cryopreserved tissue with 88.51 ± 5.93% (mean ± SD) of the isolated follicles confirmed viable using LIVE/DEAD evaluation.ConclusionsOur results indicate the ovarian tissue from deceased donors maintain high quality after long time extracorporeal circulation and transportation from the hospital to the laboratory. High survival rate of follicles at different developmental stages suggested tolerance to the cryopreservation process. Human ovarian tissues obtained from deceased donors is an ample source tissue and can be applied to promoting research and future clinical applications.

Project description:IntroductionKnee osteoarthritis is a common condition that affects daily functioning and decreases the quality of life. There are many ways of treatment depending on the stage of the disease. Advanced cases are qualified for arthroplasty, which is an extensive and demanding surgical procedure. Less advanced stages are treated in various ways: from rehabilitation, through oral and intra-articular pharmacotherapy, to surgical treatment (arthroscopy, osteotomy). Because surgical treatment is risky, scientists focus on less invasive therapeutic methods. The most valuable management is based on regeneration. Mesenchymal stromal cells (MSC) derived from the adipose tissue have a great regenerative and anti-inflammatory potential, therefore an attempt is being made to take advantage of them in knee osteoarthritis treatment.The study aims to compare the clinical effects of treatment of knee osteoarthritis using adipose tissue MSC obtained by an enzymatic method with the outcomes of the therapy with the mechanically fragmented adipose tissue.MethodsOne hundred adults with primary knee osteoarthritis will undergo lipoaspiration under sterile conditions. The collected lipoaspirates will be further processed, depending on the randomly assigned group-enzymatically with the use of collagenase or mechanically using the Lipogems system. The preparations will be administered to the patients' knee joints in the operating room under ultrasound control.The results of treatment will be assessed using Knee Injury and Osteoarthritis Outcome Score, measuring the flexibility of the knee joint, evaluating joint gap in X-ray and the quality of cartilage in magnetic resonance T2-mapping during 1 year after treatment.Discussion/conclusionIdentification and functional analysis of the regenerative capacity of adipose-derived MSC depending on three variables (body weight, sex, and age) will help to develop a targeted therapy for different groups of patients and will determine the effectiveness of both methods of treatment. An attempt will be made to identify groups of patients with the greatest regenerative potential of the adipose tissue, and thus indicate those with the most probable improvement of the joint condition.Trial registrationThis study protocol has been approved by the Ethics Committee of Medical University of Warsaw and registered on www.clinicaltrials.gov: NCT04675359 (06 Jan 2021).

Dataset Information

Successful isolation of viable stem cells from cryopreserved microfragmented human adipose tissue from patients with knee osteoarthritis - a comparative study of isolation by tissue explant culture and enzymatic digestion.

Purpose

Methods

Results

Conclusions

Publications

Successful isolation of viable stem cells from cryopreserved microfragmented human adipose tissue from patients with knee osteoarthritis - a comparative study of isolation by tissue explant culture and enzymatic digestion.

Similar Datasets

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