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Published on: February 3, 2023
The Impact of Ammonium Chloride-Based Erythrocyte Lysis Process on Banked Adipose-Derived Stem Cells
Zifei Li1, Dali Mu1, Chunjun Liu1
1Breast Plastic and Reconstructive Surgery Center of Plastic Surgery Hospital, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, People's Republic of China.
Abstract:
The safety of banked human adipose-derived stem cells (hADSCs) purified by 155 mM ammonium chloride (NH4Cl)-based erythrocyte lysis has not been evaluated. This study was conducted to determine the impact of NH4Cl-based erythrocyte lysis on the biological characteristics of cryopreserved hADSCs. Stromal vascular fractions (SVFs) were obtained from lipoaspirates and purified with NH4Cl-based erythrocyte lysis (lysis group) or without (nonlysis group). The hADSCs were freshly isolated (fresh group) from SVFs and/or cryopreserved for 2 weeks (cryo group). The morphologies, immunophenotypes, viability, apoptosis, and growth kinetics of each group were compared. The cell cycle and differentiation capacity assays were performed in both cryopreserved groups. All groups showed similar cell morphology, immunological phenotypes, and viability. However, the main effect of lysis and its interaction with cryopreservation were observed when early apoptosis was regarded as a dependent variable in two-way repeated-measures analysis of variance. After cryopreservation, significant growth retardation and S-phase fraction reduction were observed in lytic hADSCs compared with those in nonlytic hADSCs. No significant differences in the adipogenic and osteogenic differentiation capacities were found between the two groups. Although NH4Cl-based erythrocyte lysis did not affect the cell morphology, immunological phenotypes, viability, and adipogenic and osteogenic differentiation capacities of cryopreserved hADSCs, exposure to NH4Cl-based erythrocyte lysis or its synergistic action with cryopreservation may induce apoptosis and inhibit the proliferation and mitosis of cryopreserved hADSCs. These results indicate that NH4Cl-based erythrocyte lysis is not suitable for high-quality banked collection of hADSCs for future clinical applications. Further development of safe, convenient, and cost-effective purification methods of hADSCs is warranted.
Insights
Ammonium chloride lysis impacts banked human adipose-derived stem cells (hADSCs), increasing apoptosis and inhibiting proliferation after cryopreservation. This method is unsuitable for clinical applications, necessitating safer purification techniques for hADSCs.
Area of Science:
- Stem Cell Biology
- Cellular Therapeutics
- Biotechnology
Background:
- Banked human adipose-derived stem cells (hADSCs) are crucial for regenerative medicine.
- Erythrocyte lysis is a common purification step, but its impact on hADSCs is not fully understood.
- Ammonium chloride (NH4Cl) is frequently used for erythrocyte lysis.
Purpose of the Study:
- To evaluate the safety and biological impact of NH4Cl-based erythrocyte lysis on cryopreserved hADSCs.
- To determine if NH4Cl lysis affects hADSC morphology, immunophenotype, viability, apoptosis, and proliferation.
- To assess the effect of NH4Cl lysis on the differentiation potential of cryopreserved hADSCs.
Main Methods:
- Stromal vascular fractions (SVFs) were isolated from lipoaspirates.
- SVFs were purified using NH4Cl-based erythrocyte lysis or without lysis.
- hADSCs were freshly isolated or cryopreserved, and their characteristics were compared across groups.
Main Results:
- NH4Cl lysis did not alter cell morphology, immunophenotype, or viability.
- Lysis significantly increased early apoptosis, especially when combined with cryopreservation.
- Cryopreserved hADSCs purified with NH4Cl showed growth retardation and reduced S-phase fraction.
Conclusions:
- NH4Cl-based erythrocyte lysis is not suitable for banking hADSCs for clinical use due to induced apoptosis and inhibited proliferation.
- The synergistic effect of NH4Cl lysis and cryopreservation negatively impacts hADSC quality.
- Development of alternative, safe, and cost-effective purification methods for hADSCs is essential.
