Processing and Ex Vivo Expansion of Adipose Tissue-Derived Mesenchymal Stem/Stromal Cells for the Development of an Advanced Therapy Medicinal Product for use in Humans
Abstract
:1. Introduction
2. Materials and Methods
2.1. Lipoaspirate Collection
2.2. Isolation of SVF Cells
2.3. Modification of SVF Isolation Procedure by Adding the Step of Red Blood Cell Lysis
2.4. Cell Counting and Viability Assessment
2.4.1. RD Phase
2.4.2. Implementation Phase
2.5. Culture of AT-MSCs
2.6. Kinetics of AT-MSCs Growth
2.7. Antigenic Phenotyping by Flow Cytometry
2.8. Trilineage Differentiation of AT-MSCs
2.9. Chondrogenic Differentiation of AT-MSCs in Microenvironment Resembling Conditions in Human Joints
2.10. Histochemical Staining
2.11. Validation of Manufacturing Process of HE-ATMP
2.12. Stability Study of HE-ATMP
2.13. Sterility Testing
2.14. Endotoxins’ Testing
2.15. Statistical Analysis
3. Results
3.1. SVF Cells Were Effectively Isolated from Adipose Tissue
3.2. AT-MSCs Were Efficiently Cultured in In Vitro Conditions in Accordance with GMP Requirements
3.3. The Identity of Isolated AT-MSCs Was Confirmed According to ISCT Recommendations
3.4. Validation of Manufacturing Process of HE-ATMP
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Stage I: Isolation of SVF Cells | |||
Parameter | Correct Result | Incorrect Result | Method |
(1) Cell number | Presence of SVF cells (successful isolation) | Lack of SVF cells (unsuccessful isolation) | Propidium iodide staining method combined with advanced image analysis (ADAM-MCTM automated cell counter) |
(2) Cell viability | ≥60% → seeding SVF cells at a density 5000–11,000 cells/cm2 | <60% → decrease in seeding growth area | Propidium iodide staining method combined with advanced image analysis (ADAM-MCTM automated cell counter) |
(3) Sterility of the starting material | Sterile → culture of cells | Non-sterile → utilization of cell culture | Direct inoculation (BD Bactec FX400 system) |
Stage II: Culture of AT-MSCs | |||
Parameter | Correct Result | Incorrect Result | Method |
(1) Potential signs of infection of cell culture | Orange or red-raspberry clear culture medium → culture of cells | Yellow and/or turbid culture medium → evaluation of cell morphology | Macroscopic observation, light microscopy |
(2) Cell morphology | Characteristic for MSCs (spindle-shaped, elongated cells possessing fibroblast-like morphology) | Morphology different than characteristic for MSCs → utilization of cell culture | Light microscopy |
(3) Confluence of cells | ≥60% → passage of AT-MSCs <60% → further cell culture | <10% → evaluation of presence of self-detached cells | Light microscopy |
(4) Presence of self-detached cells (qualitative assessment) | Lack of self-detached cells | Presence of a high amount of self-detached cells → utilization of cell culture | Light microscopy |
(5) Cell number | At least 2× higher than number of seeding cells → continuation of manufacturing process | Less than 2× higher than number of seeding cells → utilization of cells | Propidium iodide staining method combined with advanced image analysis (ADAM-MCTM automated cell counter) |
(6) Cell viability | ≥70% → continuation of the manufacturing process | <70% → utilization of cell culture | Propidium iodide staining method combined with advanced image analysis (ADAM-MCTM automated cell counter) |
Stage III: Termination of AT-MSCs Culture and Formulation of the Final Product | |||
Parameter | Correct Result | Incorrect Result | Method |
Parameters (1)–(4) from Stage II: Culture of AT-MSCs | |||
Cell number | 12 × 106 viable cells | >12 × 106 viable cells → the final product is not released | Propidium iodide staining method combined with advanced image analysis (ADAM-MCTM automated cell counter) |
Cell viability | ≥70% → preparation of the final product | <70% → the final product is not released | Propidium iodide staining method combined with advanced image analysis (ADAM-MCTM automated cell counter) |
Specification of the HE-ATMP | |||
---|---|---|---|
Parameter | Limit or Range | Method | Additional Information |
(1) Cell morphology | Characteristic for MSCs (spindle-shaped, elongated cells possessing fibroblast-like morphology) | Light microscopy | Parameter tested before the last passage |
(2) Endotoxins | <2.5 UI/mL | LAL method (Endosafe®-PTS™ system) | Parameter tested before the last passage; the product is released before completion of the test |
(3) Sterility of the final product | Sterile | Direct inoculation (BD Bactec FX400 system) | Sample is collected at the last passage and after the final product formulation; because of the short life-time of the product, the product is released before completion of the test |
(4) Cell number per dose | 10 × 106 viable cells | Propidium iodide staining method combined with advanced image analysis (ADAM-MCTM automated cell counter) | Parameter tested after the last passage |
(5) Cell viability | ≥70% | Propidium iodide staining method combined with advanced image analysis (ADAM-MCTM automated cell counter) | Parameter tested after the last passage |
Validation of Manufacturing Process of HE-ATMP | ||
---|---|---|
Parameter | Limit or Range | Results |
(1) Cell morphology | Characteristic for MSCs (spindle-shaped, elongated cells possessing fibroblast-like morphology) | Batch no. 1: Correct |
Batch no. 2: Correct | ||
Batch no. 3: Correct | ||
(2) Endotoxins | <2.5 UI/mL | Batch no. 1: Correct |
Batch no. 2: Correct | ||
Batch no. 3: Correct | ||
(3) Sterility of the final product | Sterile | Batch no. 1: Sterile |
Batch no. 2: Sterile | ||
Batch no. 3: Sterile | ||
(4) Cell number per dose | 10 × 106 viable cells | Batch no. 1: 10 × 106 viable cells |
Batch no. 2: 10 × 106 viable cells | ||
Batch no. 3: 10 × 106 viable cells | ||
(5) Cell viability | ≥70% | Batch no. 1: 98% |
Batch no. 2: 96% | ||
Batch no. 3: 98% |
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Labedz-Maslowska, A.; Szkaradek, A.; Mierzwinski, T.; Madeja, Z.; Zuba-Surma, E. Processing and Ex Vivo Expansion of Adipose Tissue-Derived Mesenchymal Stem/Stromal Cells for the Development of an Advanced Therapy Medicinal Product for use in Humans. Cells 2021, 10, 1908. https://doi.org/10.3390/cells10081908
Labedz-Maslowska A, Szkaradek A, Mierzwinski T, Madeja Z, Zuba-Surma E. Processing and Ex Vivo Expansion of Adipose Tissue-Derived Mesenchymal Stem/Stromal Cells for the Development of an Advanced Therapy Medicinal Product for use in Humans. Cells. 2021; 10(8):1908. https://doi.org/10.3390/cells10081908
Chicago/Turabian StyleLabedz-Maslowska, Anna, Agnieszka Szkaradek, Tomasz Mierzwinski, Zbigniew Madeja, and Ewa Zuba-Surma. 2021. "Processing and Ex Vivo Expansion of Adipose Tissue-Derived Mesenchymal Stem/Stromal Cells for the Development of an Advanced Therapy Medicinal Product for use in Humans" Cells 10, no. 8: 1908. https://doi.org/10.3390/cells10081908
APA StyleLabedz-Maslowska, A., Szkaradek, A., Mierzwinski, T., Madeja, Z., & Zuba-Surma, E. (2021). Processing and Ex Vivo Expansion of Adipose Tissue-Derived Mesenchymal Stem/Stromal Cells for the Development of an Advanced Therapy Medicinal Product for use in Humans. Cells, 10(8), 1908. https://doi.org/10.3390/cells10081908