Examination of Optimal Transport Conditions for Adipose‐Derived Mesenchymal Stem Cells for Clinical Cell Transport

Background: Efficient transport of mesenchymal stem cell (MSC) products represents a major practical barrier to the clinical implementation of regenerative medicine. Clinically approved intravenous preservation fluids are not optimised for MSC preservation, and many standard hypothermic preservation solutions are not approved for direct injection, necessitating an additional washing step prior to administration. Additionally, maintaining the immunophenotypic identity of MSCs—as defined by the International Society for Cell and Gene Therapy (ISCT) minimal criteria—during transport is essential to confirm that clinically significant cellular characteristics are preserved. Methods: Human adipose-derived MSCs were suspended in normal saline, 5% dextran L (a Japanese-approved injectable formulation), and Lactated Ringer's solution. Samples were stored at refrigerated (4°C), room temperature (25°C), and body temperature (37°C) conditions. Viability was determined by trypan blue exclusion using an automated cell counter at hourly intervals from 0 to 4 hours. Immunophenotypic characterisation by flow cytometry (FCM) was performed on MSCs stored in the optimal (4°C dextran L) and worst (37°C normal saline) conditions at both 0 hours (pre-storage baseline) and 4 hours to assess preservation of ISCT surface marker criteria. Cellular membrane integrity at both timepoints was additionally confirmed by 7-aminoactinomycin D (7-AAD) exclusion staining. Results: Viability was not markedly affected across solutions and temperatures up to 2 hours; however, viability declined from 3 hours onwards. Storage at 37°C had a pronounced detrimental effect. FCM analysis confirmed full ISCT compliance in 4°C dextran L at 4 hours: CD73⁺ (99.96%), CD105⁺ (99.98%), CD90⁺ (99.47%), CD34⁻ (0.08%), CD45⁻ (0.003%), and HLA-DR⁻ (0.61%). In contrast, MSCs stored in 37°C normal saline for 4 hours exhibited two ISCT violations: CD105+ expression fell to 92.51% (below the ≥95% threshold) and CD34− expression rose to 12.49% (above the ≤2% threshold). 7-AAD staining confirmed near-complete membrane integrity at baseline in both conditions (0.000% and 0.061% 7-AAD⁺, respectively). In 4°C dextran L, 7-AAD positivity remained at 0.000% at 4 hours. In 37°C normal saline, 7-AAD positivity was unchanged at 0.061% at 4 hours despite a 49-percentage-point trypan blue viability decline, indicating that cellular deterioration under warm-storage conditions occurs predominantly through sub-lytic membrane compromise not captured by 7-AAD exclusion alone. Conclusions: Cell death begins within 2–3 hours following suspension in clinically approved solutions, indicating that transport conditions critically affect viability. MSCs stored in 4°C dextran L maintain full ISCT-compliant immunophenotypic profile at 4 hours with 100% 7-AAD-confirmed membrane integrity, suggesting that dextran L preserves both phenotypic identity and structural integrity beyond the limits detectable by trypan blue alone. Critically, MSCs stored in 37°C normal saline for 4 hours demonstrated two ISCT violations—CD34 upregulation (12.49%) and reduced CD105+ expression (92.51%)—alongside a marked trypan blue viability decline (Δ ≈ −49 pp) without a corresponding 7-AAD increase. This discordance indicates that warm-storage cellular deterioration proceeds predominantly via sub-lytic stress mechanisms, underscoring the complementary value of trypan blue and 7-AAD for MSC product quality control. Furthermore, cells replated after 4-hour storage in 4°C dextran L demonstrated normal plastic adherence at 24 hours post-replating, confirming preservation of this fundamental MSC functional property. Dextran L offers the practical advantage of obviating a post-storage wash step and may represent a pragmatic transport medium for clinical MSC workflows.