Reconstitution and post-thaw storage of cryopreserved human mesenchymal stromal cells: Pitfalls and optimizations for clinically compatible formulants

Abstract

Introduction: The regenerative and immunomodulatory properties of multipotent mesenchymal stromal cells (MSCs) make them an intriguing asset for therapeutic applications. An off-the-shelf approach, using pre-expanded cryopreserved allogenic MSCs, bypasses many practical difficulties of cellular therapy. Reconstitution of a MSC product away from cytotoxic cryoprotectants towards a preferred administration solution might be favorable for several indications. Variations in MSC handling accompanied by a non-standardized use of reconstitution solutions complicate a general clinical standardization of MSC cellular therapies. In this study, we aimed to identify a simple and clinically compatible approach for thawing, reconstitution, and post-thaw storage of cryopreserved MSCs.

Methods: Human adipose tissue-derived MSCs were expanded in human platelet lysate (hPL) supplemented culture medium and cryopreserved using a dimethyl sulfoxide (DMSO)-based cryoprotectant. Isotonic solutions (saline, Ringer's acetate and phosphate buffered saline (PBS)) with or without 2% human serum albumin (HSA) were used as thawing, reconstitution, and storage solutions. MSCs were reconstituted to 5 × 10⁶ MSCs/mL for evaluating MSC stability. Total MSC numbers and viability were determined using 7-aminoactinomycin D (7-AAD) and flow cytometry.

Results: For thawing cryopreserved MSCs the presence of protein was proven to be essential. Up to 50% of MSCs were lost when protein-free thawing solutions were used. Reconstitution and post-thaw storage of MSCs in culture medium and widely used PBS demonstrated poor MSC stability (>40% cell loss) and viability (<80%) after 1 h of storage at room temperature. Reconstitution in simple isotonic saline appeared to be a good alternative for post-thaw storage, ensuring >90% viability with no observed cell loss for at least 4 h. Reconstitution of MSCs to low concentrations was identified as critical. Diluting MSCs to <10⁵/mL in protein-free vehicles resulted in instant cell loss (>40% cell loss) and lower viability (<80%). Addition of clinical grade HSA could prevent cell loss during thawing and dilution.

Conclusion: This study identified a clinically compatible method for MSC thawing and reconstitution that ensures high MSC yield, viability, and stability. The strength of the method lies within the simplicity of implementation which offers an accessible way to streamline MSC therapies across different laboratories and clinical trials, improving standardization in this field.

Keywords: Cell viability; Cellular therapy; Cryopreservation; Mesenchymal stromal cells; Reconstitution; Thawing.

Fig. 5

Effect of temperature and used diluent on MSC stability at a low cell concentration (1 × 10⁵ cells/mL). (A, C) Percentage recovery (total cells after dilution compared to total cells prior to dilution) after dilution of thawed MSCs and freshly harvested MSCs. (B, D) Percentage of dead cells (7-AAD positive) after dilution of thawed MSCs and freshly harvested MSCs. Data represents the mean ± standard deviation of five independent experiments using five MSC batches derived from different donors ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001 versus dilution in culture medium (room temperature) by one-way ANOVA.

Fig. 7

Thawing, dilution, and storage of MSCs using clinical compatible solutions (saline and saline + HSA). (A) Percentage recovery (total cells after dilution compared to total cells prior to dilution) after thawing in saline-HSA. (B) Percentage of dead cells (7-AAD positive) after thawing in saline-HSA. (C) Percentage recovery (total cells after dilution compared to total cells prior to dilution) after dilution in saline-HSA. (D) Percentage of dead cells (7-AAD positive) after dilution in saline-HSA. (E, F) Total cell amount and percentage of dead cells (7-AAD positive) after 4 h of storage in plain saline or saline-HSA (after thawing in saline-HSA). Data represents the mean ± standard deviation of five independent experiments using five MSC batches derived from different donors. ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001 versus culture medium by one-way ANOVA.

Fig. 3

Light microscopy pictures of MSCs stored on wet ice or at room temperature in different storage solutions. Cell aggregates (arrows) are observed during storage at room temperature in culture medium and in PBS. Images acquired at × 40 magnification.

Fig. 6

Effect of the presence of supporting cells on dilution induced MSC loss. (A) Representative scatter plots of PKH26 prelabeled MSCs (PKH-MSCs) diluted in culture medium, Ringer's acetate, or Ringer's acetate supplemented with 2.5 × 10⁶/mL unlabeled MSCs. (B) Percentage MSC recovery (total cells after dilution compared to total cells prior to dilution) after dilution in the presence and absence of supporting cells. Data represents the mean ± standard deviation of three independent experiments using three MSC batches derived from different donors. ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001 versus dilution in culture medium by one-way ANOVA.

Fig. 1

Effects of different thawing solutions on MSC stability. (A) Percentage recovery (total cells after thawing compared to total cells prior to thawing). (B) Percentage of dead cells (7-AAD positive). Data represents the mean ± standard deviation of five independent experiments using five MSC batches derived from different donors. ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001 versus culture medium (in thawing solution) by one-way ANOVA. ^#p <0.05, ^##p <0.01, ^###p <0.001 versus culture medium (after reconstitution) by one-way ANOVA.

Fig. 2

Effects of different storage solutions and temperature on MSC stability over 4 h. (A) Total cell amount during storage when stored on wet ice or at room temperature. (B) Percentage of dead cells (7-AAD positive) during storage when stored on wet ice or at room temperature. Data represents the mean ± standard deviation of five independent experiments using five MSC batches derived from different donors.

Fig. 4

Effect of MSC concentration on MSC stability. (A) Total cell amount after dilution in Ringer's acetate at baseline and after 2 h of storage. (B) Percentage of dead cells (7-AAD positive) after dilution in Ringer's acetate at baseline and after 2 h of storage. Data represents the mean ± standard deviation of five independent experiments using five MSC batches derived from different donors. ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001 versus 5 × 106 cells/mL samples (baseline) by two-way ANOVA.