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Unveiling the phenotypic impact of cryopreservation on adipose derived stem cells: A systematic review
Present address: †Centre for Global Health Research, Saveetha Medical College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Chennai, Tamil Nadu, 600077, India
For correspondence: Dr Nur Azida Mohd Nasir, Plastic and Reconstructive Sciences Unit, School of Medical Sciences, Health Campus, Universiti Sains Malaysia, Kubang Kerian, 161 50, Kelantan, Malaysia e-mail: nurazida@usm.my
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Received: ,
Accepted: ,
Abstract
Background & objectives
The impact of cell passaging and cryopreservation on the phenotypic and functional attributes of adipose-derived stem cells (ADSCs) must be understood to improve their clinical value. This systematic review investigates the phenotypic characteristics of ADSCs derived from fresh and cryopreserved adipose tissue, with a focus on how these cells change across passages.
Methods
A thorough search of databases was conducted as per the PRISMA guideline to find publications that were aligned with the inclusion criteria and were published between January 2013 and January 2023.
Results
In both cryopreserved and fresh stromal vascular fraction (SVF) cells, CD90, CD73, and CD105 consistently exhibited strong expression (90%) across passages in 50 screened studies. In fresh tissue, CD29 was upregulated in subsequent passages (up to 95%) but downregulated at passage 2 (2.3%). Variable CD29 was seen in cryopreserved groups (47% at P1, 90% at P4). While CD34 and CD45 were lower in cryopreserved ADSCs (less than 5%), they were higher in fresh tissue (41%), suggesting less haematopoietic contamination.
Interpretation & conclusions
Passaging and cryopreservation protocols can help maintain the therapeutic potential of ADSCs, providing a reliable source of functional stem cells for regenerative utilization.
Keywords
Adipose-derived stem cells
cluster of differentiation
immunophenotypes
cryopreservation
systematic review
Due to rising obesity rates, adipose tissue, which contains mesenchymal stem cells (MSCs),is easily accessible. However, bone marrow remains a conventional but constrained source that frequently produces insufficient amounts of MSCs1,2. Adipose-derived stem cells (ADSCs) are MSCs that are derived from adipose tissue and yield about 50 times as many stem cells per gram as bone marrow, making ADSCs a highly abundant MSCs’ source3. Adipose tissue has also gained recognition as a substitute source of multipotent stromal or stem cells to promote regenerative therapies due to the multilineage potential of ADSCs4. These cells are primarily isolated from the stromal vascular fraction (SVF) of adipose tissue, which contains several types of stromal, endothelial, immunological, and progenitor cells, as well5. Due to the high heterogeneity of freshly isolated ADSCs, in vitro passages are necessary before clinical implementation6,7. Cryopreservation is commonly employed to preserve ADSCs during extended culture periods and reduce contamination risks. Although research indicates that freezing ADSCs for an extended time does not result in any chromosome abnormalities, it has been documented that freezing ADSCs can affect their phenotypic traits during culture, which can induce functional degradation3,8-10.
One of the most popular techniques for the phenotypic identification of ADSCs is flow cytometry, which allows for efficient, detailed characterization of cells from heterogeneous tissue6,11. This analytical method identifies clusters of antigen-dependent antibodies where the antigens are mostly transmembrane proteins or glycoproteins, found within or on the surface of the phospholipid bilayer of the cell, hence the term cluster of differentiation (CD), and the process is called immunophenotyping12. To identify the CD markers of SVF cells, different phases of their lineage-specific differentiation, or levels of activation or inactivation, are reflected in the expression of CD molecules on their cell surfaces. Since the tissue repair capabilities of ADSCs are mainly based on their multi-lineage differentiation potential, it becomes crucial to determine whether cells derived from fresh and cryopreserved adipose tissue retain their characteristic stem cell markers, stable differentiation capacity, and paracrine functions while passaging. Therefore, a comprehensive comparison of ADSC phenotypes during in vitro passaging is warranted, as this may help elucidate potential phenotypic alterations between freshly isolated SVF and SVF obtained from cryopreserved adipose tissue.
Materials & Methods
We conducted this systematic review following the PRISMA guideline13 and PROSPERO registration was obtained for the protocol (CRD42023388349).
Eligibility criteria
This systematic review aimed to assess variations in immunophenotypes of ADSCs across different cell culture passages in both cryopreserved and non-cryopreserved or fresh adipose tissue, utilising the PICOS approach (PRISMA-P 2016) (Figure). The study population focused on human ADSCs, with an intervention comparing the immunophenotypic profiles across different passage numbers. The comparison group assessed the immunophenotype of ADSCs from fresh and cryopreserved adipose tissue samples across subsequent passages, aiming to evaluate differences in marker expression. The study design was limited to in vitro analyses, enabling a controlled comparison of immunophenotypic outcomes.
- PRISMA flow diagram depicting the article selection process across different databases, including identification, screening, eligibility, and inclusion phases. This figure was created using the PRISMA flow diagram (https://www.prisma-statement.org/prisma-2020).
An extensive search was conducted to gather relevant studies on the expression of immunophenotypes in ADSCs at various passages. The study period spanned from January 2013 to January 2023, during which relevant articles were identified, screened, and selected for inclusion in this systematic review. Cochrane, PubMed, and Scopus databases were searched systematically using predetermined keywords. Additionally, ScienceDirect and Google Scholar were searched separately to ensure comprehensive article screening, and duplicate records were removed prior to screening. The database search included MeSH keywords and employed search operators ‘OR’ and ‘AND’ to refine results (supplementary material). Keywords utilised in the search included ‘adipose tissue cells/human mesenchymal cells’, ‘autologous adipose SVF’, ‘adipose tissue-derived mesenchymal stem cell’, ‘multipotent mesenchymal stromal cells’, ‘mesenchymal cells’, ‘stromal cell’, and ‘cell line/primary cell cultures phenotype analysis/CD analysis’.
A manual search using additional terms, such as ‘stromal cell antigen analysis’, ‘adipose tissue-derived mesenchymal stem cell immunophenotyping characteristics’, and ‘adipose tissue cells CD analysis’, was also conducted, used for cross-verification across databases. The eligibility criteria focused on selecting studies that specifically analysed ADSCs’ immunophenotypic markers, using the PICOS method to guide inclusion and exclusion criteria. The publication window was restricted to the past ten years to observe recent trends and advancements.
Screening and data extraction
The final database search was completed on February 1, 2023, and disagreements during the selection process were resolved with input from a third reviewer. Research that was published with full-text in vitro studies, featured immunophenotypic characterisation of human adipose-derived stem cells in primary cells or cell lines, and was available in any language with an English translation was considered for inclusion. Exclusion criteria for the studies included not being fully accessible, using animal-derived stem cells or stem cells from non-adipose tissues, or cryopreserved ADSCs without immunophenotypic analysis. Title and abstract screenings were conducted by two reviewers, who subsequently reviewed relevant articles in full. A third reviewer assisted with any uncertainties. Finally, data analysis was performed by four separate authors to minimise bias, with key findings summarised to provide a comprehensive overview of the studies reviewed.
Results
This review initially pooled 1,457 retrieved records, and after screening and eligibility assessment, 50 studies were finally included and analysed, published over the past decade (2013-2023), focused on the immunophenotypic characteristics of ADSCs.
Donor characteristics
Total 20 studies involved female donors6,14-33, 18 included both genders2,10,34-49, and 12 studies did not specified gender50-61. The age range of the donors was 10-74 yr old (Table)2,6,10,14-30,32-61. Samples were generally prepared by washing tissues or cells to remove debris and unwanted layers, followed by centrifugation.
| Author, yr | Gender & age (yr) | Tissue / Cell type | Passage (n) / Cell count | Cryoprotectant & storage | CD markers analysed | Key outcomes |
|---|---|---|---|---|---|---|
| Thitilertdecha et al6, 2020 | Female, 47-62 | SVF (fresh, frozen) | P0 (fresh/frozen), P3-P14 (cultured)/NA | Not specified | 34, 45, 31, 90, 73, 13, 44, 29, 166, 10, HLA-ABC, HLA-DR, 106, 36, 146, 235, 144, 11b, 11c, 14, 105 | CD90 & CD73 highly expressed in all; CD13, CD105, CD44, CD29, CD166, CD10 highly expressed in fresh/thawed; HLA-ABC dim in cultured group; CD34 high and CD105, CD29, CD166, CD146, CD106 dim in fresh/thawed; some hematopoietic/endothelial markers positive across groups. |
| Minonzio et al14, 2014 | Female, 18-53 | Fresh AT/ADSCs (fresh, frozen) | 0/5×10⁵ | 20% DMSO, -180°C, LN₂ | CD34⁺, CD146⁻, CD45⁻ | 26.44% CD34⁺; CD45⁻/CD146⁻ confirmed stemness |
| Zhao et al25, 2020 | Females, 25-35 | ADSCs (fresh, frozen) | P4/NA | Not specified | CD34-PE, CD45-FITC, CD73-PE, CD105-FITC | CD34 (1.3%) and CD45 (6.2%) negative; CD73 (99.9%) and CD105 (99.6%) strongly positive; results consistent in in vivo co-culture with HGC-27 cells. |
| Agostini et al27, 2018 | Females, 52.4±1.6 | SVF (fresh, frozen) | P0-P3/NA | Not specified | CD13/APC, CD31/Alexa Fluor 488, CD34/BV421, CD45/PerCP, CD73/PE-Dazzle 594, CD90/BV510, CD105/PE-Cy7, CD146/PE | ADSCs identified as CD45⁻ (∼65%), CD13⁺, CD31⁻, CD34⁺, CD73⁺, CD90⁺, CD105⁻, CD146⁻ (∼94%); EPCs (∼18%) and pericytes. |
| Park et al28, 2018 | Female (1), NA | ADSCs (fresh, frozen) | P3-P6/1×10⁶ | Not specified | CD31-PE, CD34-FITC, CD44-PE, CD73-FITC, CD90-PE, CD105-PE | Cryo- ADSCs were positive for MSCs markers |
| Al-Saqi et al29, 2015 | Females, 27-42 | ADSCs (fresh, frozen) | P3 (fresh), P5 (cryo)/1×10⁶ | Not specified | CD3, CD14, CD31, CD34, CD45, CD73, CD80, CD90, CD105, HLA-I, HLA-II, C1, C2 | Cells were 95% positive for CD73, CD90, CD105, HLA-I; and <5% for CD3, CD14, CD34, CD45, CD80, HLA-II—consistent between fresh and cryopreserved groups. |
| Zeng et al30, 2013 | Female, 15 | ADSCs (fresh, frozen) | P3/1×10⁶ | Not specified | CD44, CD105, CD29, CD90, CD13 (positive); CD31, CD34, CD45, CD106 (negative); IgG control | ADSCs positively expressed CD44, CD105, CD29, CD90, CD13; and showed minimal expression of CD31, CD34, CD45, CD106—confirming MSC-like phenotype. |
| Mashiko et al32, 2018 | Female (6), 41.4 ± 5.3 | SVF (fresh, frozen) | P0/7.2-8.5×10⁵ | Not specified; cryo with & without CPA | CD31-PE, CD34-PE-Cy7, CD45-FITC | Cryopreservation significantly decreased CD45⁺ (hematopoietic cells); stem cell marker expression relatively unchanged; endothelial cells perish without CPA. |
| Zimmerlin et al33, 2013 | Female (8), NA | SVF (fresh, frozen) | P0/NA | Not specified | CD105-FITC, CD73-PE, CD146-biotin, CD14-PE-Cy5, CD33-PE-Cy5, CD235a-PE-Cy5, CD31-PE-Cy7, CD90-APC, CD34-Alexa700, CD45-APC-Cy7 | SVF comprised 59% ADSCs, 15.4% endothelial progenitors, 2% pericytes, 0.5% CD146⁺/CD34⁺ transitional cells—indicating cellular diversity for tissue regeneration. |
| Vakhshori et al15, 2018 | Female (5), 30-42 | ADSCs (frozen) | P3/5×10⁶ | Not specified | 73, 90, 105, 44; negative cocktail: 34, 11b, 19, 45, HLA-DR | ADSCs expressed MSC markers (44, 73, 90, 105) and showed low hematopoietic marker levels; no significant marker difference before vs. after cryopreservation at P3. |
| Zhang et al16, 2022 | Female, 34-42 | ADSCs (fresh, frozen) | P0/0.5-1×10⁶ | Not specified | 44-APC, 90-PE, 29-Alexa 488, 45-FITC | ADSCs expressed 29, 44, 90 and were negative for 45, confirming MSC phenotype. |
| Kaita et al17, 2019 | Female, NA | SVF (fresh) | P0/5×10⁵ | Not specified | 90, 105, 29, 34, 14, 45 | CD105, 45, and 14 strongly expressed in both fresh and frozen SVF; cryopreserved cells showed significantly lower CD90, 29, 34—except CD34 was not statistically significant. |
| Harris et al18, 2019 | Female, 28-59 | CADSC (frozen) | P0-P4 | 10% DMSO + FBS, -196°C | CD29, CD34, CD45 | CD29 downregulated at P4; CD34/CD45 negligible showing that the passages can reduce heterogenous cells of SVF that helps healthy growth of ADSCs |
| Shaik et al19, 2018 | Female, 27-57 | ADSCs (fresh, frozen) | P1-P4/1×10⁵ | Not specified | 146, 29, 44, 34, 105, 90, 31, 73, 45 (fluor-tagged); antibody cocktails used | Both fresh and frozen ADSCs showed >95% expression of 29, 90, 105, 44, 73 and <2% of hematopoietic markers 31, 34, 45, 146 across P1-P4. |
| Pachón-Peña et al20, 2016 | Female (16), NA | SVF (fresh, frozen) | P1/2×10⁵ | Not specified | 36, 49b, 147, HLA-abc, HLA-II, 90, 29, 106, 105, 73, 34, 45, 117, 184, 44 | hADSCs from obese individuals showed higher expression of 36, 106, and HLA-II vs. lean donors; these correlated with greater proliferation and migration, indicating immunophenotypic differences. |
| Kalinina et al21, 2015 | Female (10), <50 | ADSCs (fresh) | P3-P4/NA | Not specified | CD45, CD7, CD73, CD90, CD105, CD31, CD34, CD146, NG2, PDGFRβ; includes isotype controls (IgG-PE, PerCP, PC5) | ADSCs cultured in serum-free medium showed >97% viability and MSC phenotype (>99% CD105⁺, CD73⁺, CD90⁺, CD45⁻) through to passage 4. |
| Chaput et al22, 2014 | Female, 30 | AT/SVF (frozen) | NA/NA | 10% Me2SO + albumin, -80°C → LN₂ | CD34⁺, CD90⁺, CD73⁺ | Clinical protocol validated for banking |
| Qu et al23, 2020 | Female (18), 20-40 | SVF (fresh) | Not stated/NA | Not specified | 36-FITC, 34-PE, 19-ECD, 45-PC7, 3-PeCy7, 146-PE, 31-FITC, 73-PE | ASCs from abdomen and thighs expressed 44, 73, 90, 105 (>95%) more strongly than those from upper limb/waist, enhancing proliferation and graft retention. |
| Yong et al24, 2015 | Female, 25-35 | ADSCs (fresh, frozen) | P3/NA | Not specified | CD90-CD45, FITC, CD73-PE, CD105-FITC, CD44-FITC, HLA-ABC-FITC | Both fresh and cryopreserved ADSCs were positive for MSCs; negative for CD14, CD19, CD34, CD45, and HLA-DPDQDR. |
| Bae et al26, 2015 | Female (1), 12 | ADSCs (fresh, frozen) | P1/5×10⁴ | Not specified | 11b, 31, 44, 45, 73, 90, 105, HLA-DR | ∼90% of ADSCs expressed MSC markers (44, 73, 90, 105); hematopoietic markers (11b, 31, 45, HLA-DR) were negative, confirming MSC phenotype post-cryopreservation. |
| Mieczkowska et al42, 2018 | Both, 32-71 | SVF/ADSCs (frozen) | P1-P6/NA | STEM-CELLBANKER, -80°C → LN₂ | CD90⁺, CD73⁺, CD105⁺ | High MSC markers; stable immunophenotype |
| Shaik et al43, 2020 | Both (3), NA |
SVF (frozen) |
P2/NA | Frozen at -80 °C in different media | 146-Alexa Fluor 647, 29-FITC, 34-PE, 90-BV421, 31-APC-eFluor, 45-BV711 | Cryopreserved SVF maintained stemness; CD29 (81-94%) and CD90 (89-96%) were high; CD34 (34-61%) and CD45 (43-49%) moderate; CD31 and CD146 undetectable; no significant formulation differences. |
| Escobar & Chaparro44, 2016 | Both, 10-50 | ADSCs (frozen) | P4/5×10⁵ | Xeno-free cryopreservation, 10% DMSO, −196 °C | CD34/APC, CD45/RPE-Cy5, CD73/PE, CD90/APC, CD105/PE, HLA-ABC/FITC | High expression of MSCs and no expression of CD34, CD45, or HLA-DR CD90. |
| Shah et al45, 2013 | Both (12 females, 1 male), 44.3 ± 11.3 | SVF (fresh) | P0/2,000-9,000 cells | Not specified | CD29, CD105, CD45, CD34, CD44, CD73, CD90 | Freshly isolated SVF cells expressed CD29, CD105, CD44, CD73, CD90 positively; CD34 was negative. |
| Engela et al46, 2013 | Both (10 females, 7 males), 38.1-63.2 | ADSCs (fresh) | P2 & P6/NA | Not specified | CD14/PE, HLA-ABC/PE, CD90/APC, CD105/FITC, CD34/APC, CD45/FITC, CD166/PE, HLA-DR/APC-Cy7 | Low expression of CD34, CD90, CD105, CD166, and HLA-ABC. No expression of CD14, CD45, or HLA-DR. |
| Solodeev et al47, 2019 | Both, 42.8 ± 11.6 | SVF (fresh, frozen) | 0/NA | CPA/non-CPA; -80°C → LN₂ | CD29⁺, CD73⁺, CD90⁺, CD105⁺ | Viable stem cells post-thaw in both groups |
| Neubauer et al48, 2019 | Both (1 male, 2 females), 71.3 ± 2.9 | ADSCs (fresh) | P1/NA | Not specified | CD73, CD90, CD105; CD34, CD11b, CD19, CD45, HLA-DR (negative) | MSC markers positive; hematopoietic markers absent. ADSCs were effectively isolated from 3 distinct fat depots, confirming method versatility. |
| Helmy et al49, 2020 | Both (12 donors), 18-60 | ADSCs (fresh) | P1/NA | Not specified | CD3-FITC, CD14-FITC, CD19-FITC, CD34-PE, CD45-FITC/PE, CD49d-FITC, CD73-FITC, CD90-FITC, CD105-FITC, HLA-DR-FITC | ADSCs from both sources expressed MSC markers and were negative for hematopoietic markers, confirming isolation efficiency. |
| Shah et al34, 2016 | Both (4 donors), 34-72 | ADSCs (fresh) | P1/NA | Not specified | CD29, CD34, CD45, CD73, CD90, CD105; PE-IgG1 and FITC-IgG1 isotype controls | No significant difference in CD29, CD73, CD90, CD105 positivity and CD45 negativity between fresh and cryo-ADSCs; CD34 and CD45 higher in fresh (88.4%, 6.7%) vs. cryo (18.7%, 1%). |
| Irioda et al35, 2016 | Both (10 females, 2 males), NA | ADSCs (fresh) | P2/NA | Not specified | FITC-CD34, PE-CD45, PE-CD49d, PE-CD73, FITC- | Positive expression of MSC markers, with no significant expression of hematopoietic markers. |
| Zhou et al36, 2017 | Both (8), 24-54 | SVF, ADSCs (fresh) | P3/NA | Not specified | PE CD29, CD34; FITC CD45, CD90 | At P3, ADSCs showed higher expression of 29 (99.8%) and 90 (92.8%) vs. SVF (29.7%, 25.1%); lower 34 (11.2%) and 45 (1.4%) vs. SVF (7.9%, 15.8%)—indicating phenotypic shift with culture. |
| Kumar et al2, 2019 | Both (4), 38-53 | ADSCs (fresh, frozen) | P1/5×10⁴ | Long-term cryopreservation (12 yrs) | 90 (FITC), 166 (PE), STRO1 (PE/Cy7), 73 (APC), 105 (BV510), 45 (FITC), 34 (PE); also NOTCH1 (non-fluor labelled) | ADSCs retained strong expression of MSC markers and showed negligible levels of 34 and 45, preserving stemness post-cryopreservation. |
| Kokai et al37, 2017 | Both (1 female, 2 males), 17-29 | SVF (fresh) | P0/2.6×10⁶ | Not specified | 105-FITC, 73-PE, 146-biotin, 14-PE-Cy5, 31-PE-Cy7, 90-APC, 34-APC-Alexa700 | Older donors had fewer CD45⁻ and CD31⁺ cells, but more ADSCs progenitors than young (17-29 yrs), highlighting age-related stem cell variations. |
| Bellei et al38, 2017 | Both (21 females, 12 males), 16-74/50 ± 12 | ADSCs (fresh) | P2/2×10⁴ | Not specified | 105, 73, 90, 44, 19, 45, 31, 34, 29, 14, 11b, HLA-DR, 49d, 54 | ADSCs expressed MSC markers (44, 105, 73, 90) >95%; negative for hematopoietic markers (≤3%). Adhesion molecules 49d and 54 were also strongly expressed. |
| Zanata et al39, 2018 | Both, 45.75 ± 7.5 | SVF (fresh) | P0/3.04-3.41×10⁵ | Not specified | 36-FITC, 34-PE, 19-ECD, 45-PC7, 3-PeCy7, 146-PE, 31-FITC, 73-PE | Cryopreserved SVF showed increased expression of stromal/vascular markers (73, 36, 34, 146, 31) and reduced 45, suggesting loss of hematopoietic cells post-cryopreservation. |
| Roato et al10, 2016 | Both, NA | ADSCs (fresh, frozen) | P2/NA | Fresh, -80 °C, and -196 °C compared | 105-PE, 73-FITC, 44-FITC, 45-PerCP, 3-PerCP, 271-APC; isotype controls | ADSCs expressed CD105, CD44, CD73, CD271; negative for CD45 and CD3. Expression of CD105/44⁺/45⁻ & CD105/73⁺/271⁺/45⁻ was higher at -80 °C than fresh or -196 °C frozen samples. |
| Wang et al40, 2013 | Both, 52 | ADSCs (fresh) | P20/1×10⁵ | Not specified | 90-PE-Cy7, 105-PerCP, 34-APC, 45-PE, 31-PE | ∼80% adipocytes were viable; CD34⁺/31⁻ cells showed lower apoptosis/necrosis than CD105⁺/45⁻ cells & retained strong growth and differentiation post-cryopreservation. |
| Devitt et al41, 2015 | Both, 43.2 ± 9.7/range 26-62 | ADSCs (fresh) | P1/2.5×10⁵ | Not specified | 34, 45, 105 (PE-Cy7/APC) | Most ADSCs were 105⁺ & 45⁻; expression of 34 was variable. CD105 confirmed as a reliable MSC marker in ADSCs. |
| Lee et al50, 2014 | NA | ADSCs (fresh) | P3/NA | NA | CD29⁺, CD44⁺, CD73⁺, CD90⁺, CD105⁺, CD14⁻, CD34⁻, CD45⁻ | Strong MSC expression was retained |
| Guo et al51, 2022 | NA, 6 donors, 58 ± 21.1 | ADSCs (fresh) | P3-P6/1×10⁶ | Not specified | CD31-PE, CD34-FITC, CD44-PE, CD73-FITC, CD90-PE, CD105-PE, CD166-PE, HLA-ABC-FITC, HLA-DR-FITC | Cryo-thawed cells showed positive expression of MSCs markers and negative for CD31, CD34, and HLA-DR, regardless of cryo method. |
| Rogulska et al54, 2017 | NA (10 donors) | ADSCs (fresh, frozen) | P4/3×10⁵ | α-MEM + 100-300 mM sucrose as CPA | CD29/PE, CD34/FITC, CD45/PE, CD73/PE, CD90/FITC, CD105/FITC | >95% expression of CD73, CD90, CD105; <5% CD45 expression; comparable to fresh tissue; CPA concentration had no significant impact on phenotype. |
| Rodriguez et al55, 2015 | NA,3 donors, NA | ADSCs (fresh) | P1/1-2×10⁶ | Not specified | FITC-CD45, PE-CD90, CD73, CD14, CD34, HLA-DR | >98% expression of CD90, CD73, HLA-ABC; <2% expression of CD14, CD45, and HLA-DR, confirming stem cell purity post-digestion at passage 1. |
| Massiah et al56, 2021 | NA, 10 donors, NA | ADSCs (fresh, frozen) | P0/NA | Various cryoprotectants (not specified) | Anti-human CD36, PE-conjugated CD45 | 97-99% of adipocyte progenitor cells viable; no significant difference between fresh and frozen-thawed AT, regardless of the cryoprotectant used. |
| Raposio et al57, 2016 | NA | SVF (fresh) | P0/9×10⁵ | Not specified | CD31, CD34, CD45, CD73, CD90 | ADSCs preserved (CD45⁻/CD34⁺/CD31⁻), endothelial cells (CD90⁺/CD105⁺), and stromal cells (CD73⁺). |
| Svalgaard et al58, 2020 | NA | ADSCs (fresh, frozen) | NA/NA | 1-2% DMSO + pentaisomaltose | CD73⁺, CD90⁺, CD105⁺; CD45⁻, CD31⁻ | Maintained MSC profile; less toxicity |
| Wan59, 2017 | NA, 58±21.1 | SVF (fresh, frozen) | NA/3×10⁵ | Hypoxic -196°C | CD73, CD90, CD105 | Marker levels retained under hypoxia |
| Zhu et al60, 2013 | NA | SVF (fresh) | P0/5×10⁵ | Not specified | CD9, CD10, CD13, CD29, CD34, CD44, 49a-e, CD51, CD54, CD55, CD59, CD61, CD63, CD71, CD73, CD90, CD105, CD138, 140a, 1CD46, CD166, HLA-ABC, STRO-1 (positive); 11a-c, CD14, CD16, CD18, CD31 | Freshly isolated SVF cells expressed MSC markers and lacked hematopoietic/endothelial markers supporting their multipotent stromal phenotype. |
| Hamid et al61, 2018 | NA | ADSCs (fresh) | P4/10,000 | Not specified | 9-PE, 34-FITC, 117-PE, 31-PE, 44-FITC, 45-FITC, 73-FITC, 90-FITC | ADSCs strongly expressed 90 (99.9%), 73 (99.7%), 44 (99.4%), 9 (91.2%), HLA-ABC (91.2%). |
| Choudhery et al52, 2014 | NA | ADSCs (fresh) | P1/1×10⁵ | Not specified | 3 (AF), 14 (PE), 19 (APC), 34 (PE), 44 (APC), 45 (FITC), 73 (PE), 90 (AF), 105 (APC) | Both fresh and cryopreserved MSCs expressed CD44, CD73, CD90, and CD105 while lacking hematopoietic markers, demonstrating preserved MSCs |
| Vu et al53, 2015 | NA | ADSCs (fresh) | P3/NA | Not specified | CD31, CD34, CD45 | Passaging showed comparable marker retention |
SVF, stromal vascular fraction; ADSCs, adipose derived stem cells; AT, adipose tissue
Cryopreservation protocols
Cryopreserved samples ranged from fresh adipose tissue (1 to 6 mL), isolated SVF cells (5×105-2.5×106 cells/mL), and ADSCs at different passage numbers, with cell concentrations typically between 5×105 and 2×106 cells/mL per cryovial. Dimethyl sulfoxide (DMSO) was the most common cryoprotectant used, often at 5-10 per cent, sometimes combined with foetal bovine serum (FBS), Dulbecco's modified eagle medium (DMEM), or additives like trehalose or sucrose. Prior to freezing at -80°C and storing in liquid nitrogen (-196°C), a controlled-rate chilling approach (-1°C/min) was usually employed; however, six investigations documented other techniques (20%), whereas the storage temperatures were -20°C, -70°C, and -80°C39. Storage durations varied from several weeks to years, depending on the study. Thawing was typically done rapidly in a 37°C water bath, with cryoprotectants being removed with phosphate buffered saline (PBS) washing and centrifugation, ensuring the cells' viability for further applications10,24.
Passage numbers and cell densities
Table shows that the majority of studies utilised 3-5 passages, in the range of 1 × 10⁵-10⁶ cells, ensuring optimal cell viability and characteristics. Flow cytometry and Flowjo software were the predominant assay and analysis employed, allowing for precise quantification of cell surface markers. Several antibodies were utilized, conjugated with phycoerythrin (PE), fluorescein isothiocyanate (FITC), R-phycoerythrin (R-PE), fluorochrome krome orange (KrO), alexa fluor, cyanine5 (Cy5), peridinin-chlorophyll-protein (PerCP), Brilliant Violet, and allophycocyanin (APC). While negative markers like CD34, CD11b, CD19, and HLA-DR verified the non-haematopoietic nature of ADSCs, positive markers like CD29, CD44, CD73, CD90, and CD105, which indicate mesenchymal stem cell characteristics, were frequently observed. Additionally, endothelial marker CD31 was frequently assessed to validate the multipotent capabilities of ADSCs.
Immunophenotypic markers and expression trends
Overall, 31 distinct CD markers were observed from all the selected studies, with 18 markers (58% of studies) showing positive expression and 13 markers (42% of studies) showing negative expression across fresh and cryopreserved samples. The most frequently assessed positive markers included CD90 (38 studies, 76%, CD73, and CD105 (34 studies, 68%), indicating a strong representation of mesenchymal stem cell (MSC) characteristics. Additionally, CD34 and CD45 were evaluated in 35 studies (70%), suggesting their relevance in assessing progenitor and hematopoietic cell populations. Notably, CD31 (21 studies, 42%), CD29 (17 studies, 34%), CD14 (11 studies, 22%), HLA-ABC (10 studies, 20%), CD44 (8 studies, 16%), and CD49d (6 studies, 12%) were also prominent, highlighting potential immunogenic risks in therapeutic applications.
Furthermore, some other positive markers were less studied, e.g., CD36, CD117, CD147, CD271 (2%), CD3, CD10 (4%), CD13 (6%) while negative antigen markers like HLA-DR (20%), CD19, CD146 (12%), CD166 (10%) were more frequently studied than CD11b, and CD106 (8%), CD11a, CD144 (6%), CD3, CD11a, CD11c, and CD54 (4%). Only one study has been reported for CD49b, CD36, CD117, CD140a, and STRO-1. This effectively conveys that the differences in marker expression were related to the distinct methodologies or objectives of the studies.
In this study, the expression of CD markers across fresh adipose tissue (FAT), cryopreserved adipose tissue (CAT), cryopreserved stromal vascular fraction (CSVF), and cryopreserved adipose-derived stem cells (CADSCs) were analysed across multiple passages. CD29 had high expression in FAT at passage 0 (98%) and CADSCs at passage 0 (99%). FAT maintained high expression through passage 4 (94%), while CADSCs showed a slight decrease by passage 4 (91%).
In contrast, CAT and CSVF did not show significant CD29 expression in any passage. CD90 also showed high expression in FAT and CADSCs at passage 0 (99.8% in FAT and 95% in CADSCs). FAT maintained consistently high levels through passage 4 (98%), while CADSCs exhibited a slight reduction to 91 per cent by passage 4. For CD34, FAT had moderate expression at passage 0 (73%) but gradually decreased in later passages, showing no expression by passage 4. CSVF exhibited higher CD34 expression at passage 0 (76%) compared to CADSCs (22%), but both showed reduced levels by passage 2 (14% in CADSCs) and near complete loss by passage 4. CD45 had minimal or no expression across all groups and passages, with FAT showing <5 per cent expression at passage 2 and CADSCs exhibiting only slight expression at passage 1. CAT and CSVF did not show significant expression of CD45 at any passage (Table).
In terms of high expression, FAT and CADSCs consistently showed high levels of CD29 and CD90 throughout all passages, with only slight reductions in CADSCs after passage 2. Medium expression of CD34 was observed in CSVF at passage 0 (76%) compared to the lower expression in CADSCs (22%) at the same passage, and by passage 2, both groups exhibited low levels, with CADSCs showing 14 per cent. By passage 4, both CADSCs and CSVF showed no expression of CD34. Low or no expression of CD45 was observed consistently across all groups, with minor exceptions in early passages of FAT and CADSCs. These findings underscore that FAT and CADSCs maintained the most robust marker expression, while CAT and CSVF showed minimal or no expression across all passages.
Discussion
Our findings reflect the diversity of focus in ADSCs research, influenced by the functional relevance of certain CD markers, study aims, and the availability of standardized detection protocols. Frequently studied positive MSCs’ expression, e.g., CD90, CD73, and CD105, underscores the emphasis on confirming ADSCs' mesenchymal origin and multipotency, explaining their prominence across studies. Additionally, markers like CD34 and CD45, traditionally associated with progenitor and hematopoietic cells, are commonly assessed to distinguish ADSCs from other cell types, further justifying their frequent evaluation. In contrast, less-studied markers like CD36, CD117, CD271, and CD147 appear to play more specialised roles in angiogenesis, stemness, or immune modulation, which may not be the primary focus of all research. Negative markers like HLA-DR and CD146, often utilized to exclude immune cells, were found to be more frequently analysed in studies prioritizing the immunogenicity and clinical safety of ADSCs. This variability in marker selection reflects application-driven research priorities.
This study sheds light on how CD markers are expressed across fresh adipose tissue, cryopreserved adipose tissue, cryopreserved stromal vascular fraction, and cryopreserved adipose-derived stem cells at various passages, aiming to relate these expression levels to the quality of adipose tissue during passaging and cryopreservation. A significant finding of this research is the consistently high expression of MSC markers in both fresh adipose tissue and cryopreservation groups throughout passage 6, indicating the consistent quality of the adipose tissue, reflecting its capacity for self-renewal and differentiation even after cryopreservation41,62. One study reported that ADSCs from the fresh adipose tissue can express 90 per cent till passage 3017. However, the passaging of CAT and CSVF groups was studied less, highlighting the necessity for further exploration of these less-studied groups to fully understand the implications of cryopreservation on ADSC functionality.
CD45 expression was consistently low or absent across all groups, with a notable observation that fresh adipose tissue exhibited slightly higher expression compared to cryopreserved adipose tissue, cryo-stromal vascular fraction, and cryopreserved adipose-derived stem cells. Specifically, only 33 per cent of studies found CD45 expression in fresh adipose tissue, while cryopreserved adipose tissue and cryo-stromal vascular fraction showed minimal expression, indicating a significant reduction after cryopreservation. This pattern confirms that mesenchymal stem cells, not hematopoietic cells, make up the majority of the cell population in stromal vascular fraction of adipose tissue, supporting the non-hematopoietic origin of ADSCs. The observed decrease in CD45 expression following cryopreservation further suggests that the cryopreservation process may lead to the loss of hematopoietic cells.
CD29, also known as integrin beta-1, is a key MSC marker involved in cell adhesion, migration, and differentiation. Its stable expression through passaging in both fresh and cryopreserved adipose tissue highlights the role of passaging in maintaining ADSC purity, although cryopreserved cells may show slightly lower expression, emphasizing the need for further studies on this marker due to due to the limited existing research. In contrast, markers such as HLA-ABC, CD44, CD36, CD3, CD13, CD49d, and CD147 remain underexplored, and investigating them could provide additional insights into ADSC biology and clinical potential. However, for a thorough grasp of the biology of ADSCs and their clinical relevance, it is imperative to investigate these markers.
The expression of CD markers across adipose tissue, SVF, and ADSCs were found to be varied by differences in cryopreservation techniques, such as cryoprotectants, cooling rates, and storage times, particularly over many passages. According to studies, the type of cryoprotectant used has a significant impact on post-thaw cell viability; commercial solutions like STEM-CELLBANKER or combinations like dimethyl sulfoxide and foetal bovine serum are effective.
Additionally, slower cooling rates may enhance the survival of adipose-derived cells, while prolonged storage can lead to reduced expression of key CD markers. Moreover, the observed variability in CD marker expression, especially with markers like CD44, CD10, and CD13, might be attributed to differences in cryopreservation parameters. Fresh samples tended to maintain more consistent expression profiles, whereas cryopreserved samples show discrepancies; for instance, CD166 is not expressed in cryopreserved SVF but showed high expression in cryopreserved ADSCs. This suggests a possible correlation between cryopreservation protocols and CD expression stability, particularly as cells were passaged in vitro.
As a mechanistic consideration, several molecular pathways might be responsible for the downregulation or deletion of surface markers like CD29 and CD34 after cryopreservation. Oxidative stress brought on by cryopreservation can harm membrane-bound proteins and cause lipid peroxidation. Furthermore, freeze-thaw damage may selectively decrease the survival of specific subpopulations by activating apoptotic signalling pathways. Genes producing surface indicators may potentially be silenced by epigenetic drift, including changes in DNA methylation and histone modification, especially after prolonged storage or repeated passaging. The phenotypic instability seen in several studies was probably caused by these mechanisms.
Therapeutic implications of CD marker expression
Although the International Society for Cellular Therapy (ISCT) suggests that MSCs express ≥95 per cent of CD73, CD90, and CD105 and ≤2 per cent of CD45 and CD34, there is no agreed-upon cutoff point for therapeutic effectiveness. Numerous in vivo investigations have shown positive results even in cases when marker expression does not meet these requirements. For instance, in a model of pulmonary fibrosis, CD73⁺ ADSCs have been shown to decrease fibrosis and macrophage infiltration63. Additionally, in clinical applications, CD31⁺ ADRCs have shown greater paracrine and angiogenic effects, which correlate with improved erectile performance64. Furthermore, the resultant cells maintained their therapeutic effect even when CD105 expression dramatically dropped during differentiation into melanocyte-like cells65,66. These results suggest that even when surface marker expression does not precisely reach predetermined criteria, positive results can still be obtained.
As per these findings, significant clinical effects can still be linked to slower or variable marker expression, but it is undeniable that changes in the expression of CD29 and CD34 can have a major effect on vital therapeutic functions such immunomodulation, differentiation capacity, and cell homing. For instance, decreased angiogenic capacity and impaired contact with endothelial cells have been linked to downregulation or deletion of CD34, which limits the cell's ability to promote vascular regeneration67. Integrin β1, or CD29, essential for cellular adhesion, migration, and survival signalling; decreased expression may make it more difficult to migrate to wounded regions and prevent regenerative integration68.
In conclusion, the main mesenchymal markers were found to be typically retained in ADSCs following cryopreservation, according to this systematic study, particularly when cells were isolated after thawing. However, relevant cross-study comparisons are limited by significant variation in cryopreservation techniques and uneven assessment of less-characterised markers. More reproducibility and clinical translation can be achieved with functional validation and established procedures. To fully use the therapeutic potential of cryopreserved ADSCs in regenerative medicine, strong, standardized procedures of cryopreservation of adipose tissue, passaging methods of ADSCs, as well as thorough phenotypic and functional evaluations, are necessary going forward.
Acknowledgment
Authors acknowledge all the staff of the Reconstructive Sciences Unit, Universiti Sains Malaysia, Malayasia.
Financial support & sponsorship
The study was supported by the Fundamental Research Grant Scheme (Grant Number: FRGS/1/2020/SKK0/USM/03/12) from the Ministry of Higher Education, Malaysia.
Conflicts of Interest
None.
Use of Artificial Intelligence (AI)-Assisted Technology for manuscript preparation
The authors confirm that there was no use of AI-assisted technology for assisting in the writing of the manuscript and no images were manipulated using AI.
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