1. Stem Cells and Regenerative Medicine

Comparison of freshly cultured versus cryopreserved mesenchymal stem cells in animal models of inflammation: A pre-clinical systematic review

  1. Chintan Dave
  2. Shirley HJ Mei
  3. Andrea McRae
  4. Christine Hum
  5. Katrina J Sullivan
  6. Josee Champagne
  7. Tim Ramsay
  8. Lauralyn McIntyre  Is a corresponding author
  1. Division of Critical Care Medicine, Department of Medicine, Western University, Canada
  2. Regenerative Medicine Program, Ottawa Hospital Research Institute, Canada
  3. Knowledge Synthesis Group, Ottawa Hospital Research Institute, Canada
  4. University of Ottawa, Canada
  5. Clinical Epidemiology, Ottawa Hospital Research Institute, Canada
  6. Division of Critical Care, Department of Medicine, University of Ottawa, Canada
https://doi.org/10.7554/eLife.75053
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Cite this article
  1. Chintan Dave
  2. Shirley HJ Mei
  3. Andrea McRae
  4. Christine Hum
  5. Katrina J Sullivan
  6. Josee Champagne
  7. Tim Ramsay
  8. Lauralyn McIntyre
(2022)
Comparison of freshly cultured versus cryopreserved mesenchymal stem cells in animal models of inflammation: A pre-clinical systematic review
eLife 11:e75053.
https://doi.org/10.7554/eLife.75053
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5 figures, 7 tables and 5 additional files

Figures

Figure 1
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Literature search and study inclusion.
Figure 2
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Primary in vivo outcomes.

All the outcomes related to function and composition of tissues are presented below. Number of experiments represent the number of separate comparisons between freshly cultured and cryopreserved MSCs on surrogate measures of in vivo efficacy.

Figure 3
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Primary in-vivo outcomes.

All the outcomes related to protein (cytokine) expression and secretion are presented below. Number of experiments represent the number of separate comparisons between freshly cultured and cryopreserved MSCs on surrogate measures of in vivo efficacy.

Figure 4
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In-vitro potency outcomes.

All the in-vitro reported outcomes are displayed below. Number of experiments represent the number of separate comparisons between freshly-cultured and cryopreserved MSCs on surrogate measures of in vivo efficacy.

Figure 5
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Comparison of viability.

Experiments where viability at varying time points of freshly-cultured and cryopreserved MSCs were compared directly are presented below.

Tables

Table 1
Models of inflammation and characteristics of included studies.
First Author (Year)Animal Inflammatory ModelCountryLanguage of PublicationSpeciesStrainGenderSample sizeAge (range)Weight (grams)
Bárcia et al., 20171) Chronic adjuvant-induced arthritis (AIA) model
2) Hindlimb ischemia model		PortugalEnglish1) Rat
2) Mouse		1) Winstar
2) C57BL/6		1) Male
2) Female		1) 18
2) 36		1) NR
2) 12 weeks		1) 365–480 g
2) NR
Cruz et al., 2015Allergic Airways Inflammation induced by Aspergillus hyphal extract (AHE) exposure in immunocompetent miceUSAEnglishMouseC57BL/6Male728–12 weeksNR
Curley et al., 2017Acute respiratory distress syndrome by intratracheal instillation of E. coliCanadaEnglishRatSprague-Dawley (specific pathogen-free)MaleNRNR350–450 g
Devaney et al., 2015Acute lung injury induced by E. coli pneumoniaIrelandEnglishRatSprague-Dawley (specific pathogen-free)Male40NR350–450 g
Gramlich et al., 2016Retinal Ischemia/Reperfusion Injury ModelUSAEnglishMouseC57BL6/JMale and Female372 monthsNR
Lohan et al., 2018Corneal TransplantationIrelandEnglishRatLewisMaleNR8–14 weeksNR
Salmenkari et al., 2019Colitis (3% DSS)FinlandEnglishMouseBalb/cMaleNR8 weeksNR
Somal et al., 2017Wound healingIndiaEnglishRatWistarMale27NRNR
Bharti et al., 2020Wound healingIndiaEnglishGuinea pigsDunkin HartleyMale25NRNR
Horie et al., 2020aVentilator-induced Lung InjuryIrelandEnglishRatsNRNRNRNRNR
Horie et al., 2020aE. coli-induced lung injuryIrelandEnglishRatsPathogen-free sprague DawleyMaleNRNR300–450 g
Khan et al., 2019Spinal Cord Injury induced through a balloon compression methodKoreaEnglishDogBeagleNR121.2+/-0.2 years12+/-3 kg
Rogulska et al., 2019Wound healingUkraineEnglishMouseBalb/CMale275–6 months25–30 g
Tan et al., 2019Polymicrobial sepsis induced by cecal-ligation-and-puncture (CLP)CanadaEnglishMouseC57BL6/JFemaleNR8 weeks17–21 g
Perlee et al., 2019K.K. pneumoniae induced pneumosepsisNetherlandsEnglishMousePathogen free C57BL/6FemaleNR8–10 weeksNR
Yea et al., 2020Wound healingKoreaEnglishRatSprague-DawleyMale12012 weeks340–360 g
Horiuchi et al., 2021OsteoarthritisJapanEnglishRatWildtype LewisFemale4010 weeks180–200 g
Horie et al., 2021Ventilator-Induced Lung InjuryIrelandEnglishRatSprague-DawleyMale28NR350–450 g

  1. NR = Not Reported.

Table 2
MSC characteristics of included studies.
First author (Year)Species and tissue sourceCompatibility with animalISCT criteria metRoute of administrationVehicleTiming of MSCs post-disease inductionFresh MSCsCryopreserved MSCs
Cryopreserved at any point?Duration of cultureMethodDurationTime from Thaw to Experiment
Bárcia et al., 2017Human Umbilical CordXenogenicYes1) Intra-articular
2) Intra-muscular		PBS1) 7 days
2) 5 hr		No>5 daysControlled Rate FreezerNRImmediately
Cruz et al., 2015Human and Murine Bone MarrowSyngenic and XenogenicYesIntravenousPBS14 daysYesNR-–80°C for 48 hr then liquid nitrogen9 days15 min
Curley et al., 2017Human Umbilical Cord and Bone MarrowXenogenicYesIntravenousPBSNRNo4 daysControlled Rate FreezerNRDay of administration
Devaney et al., 2015Human Bone MarrowXenogenicYesIntravenousPBS0.5 hrYesNRNRNR30 min
Gramlich et al., 2016HumanXenogenicYesIntra-ocularPBS2 hrYes>7 daysControlled Rate Freezer7–30 days<1 hr
Lohan et al., 2018Rat Bone MarrowAllogenicNRIntravenousPBS1 and 7 days priorYesNR–80°C for 24 hr then liquid nitrogenNRImmediately
Salmenkari et al., 2019Human Bone MarrowXenogenicNRIntravenous0.9% NaCl +3.6% HAS3 and 5 daysYesNRNRNRNR
Somal et al., 2017Gravid caprine AF (amniotic fluid), AS (amniotic sac), WJ (Wharton's jelly), CB (cord blood)XenogenicNRSubcutaneouslyPBS7, 14, 21, 28 daysYesNR–80°C overnight then liquid nitrogenAtleast 1 monthNR
Bharti et al., 2020Dog Bone MarrowXenogenicNRSurgically placed over woundPolypropylene meshNRYesNR–80°C overnight then liquid nitrogen1 monthNR
Horie et al., 2020aHuman Bone MarrowXenogenicNRIntravenousPBS6 hrYesNRNRNRNR
Horie et al., 2020aHuman Bone Marrow and Umbilical CordXenogenicNRIntra-trachealPBS30 minYesNRNRNRImmediately
Khan et al., 2019Dog Adipose TissueAllogenicNRIntravenousHartmann’s SolutionImmediatelyYesNR4 °C for 1 hr, –20 °C for 2 hr, –80 °C for 24 hr, then –150 °C2–3 weeksImmediately
Rogulska et al., 2019Human Adipose TissueXenogenicNRImplantation into wound3D gelImmediatelyYesNR–80°C the liquid nitrogen1 monthNR
Tan et al., 2019Human Bone MarrowXenogenicYesIntravenous5% Human Albumin in PlasmaLyte6 hrNo>24 hrControlled Rate FreezerNRImmediately
Perlee et al., 2019Human Adipose TissueXenogenicYesIntravenousRinger’s Lactate1 or 6 hrNo24 hrLiquid nitrogenUntil requiredDay of administration
Yea et al., 2020Human Umbilical CordXenogenicNRIntratendinousPBSImmediatelyNoNR–80°C then –196 °C Liquid NitrogenUp to 1 monthImmediately
Horiuchi et al., 2021Rat Synovial FluidAllogenicNRIntraarticularPBSEvery week from 2 to 8 weeksYes7 days–80 °C overnight, and then at –150 °C16 monthsImmediately
Horie et al., 2021Human Umbilical CordXenogenicNRIntravenousPBS15 minNoNRNRUp to 2 monthsImmediately
Table 3
Risk of Bias assessments for the included in vivo studies using SYRCLE Tool.
Selection BiasPerformance BiasDetection BiasAttrition BiasReporting BiasOther Bias
Author (year)Adequate randomizationBaseline charactersics givenEvidence of adequate concealment of groupsEvidence of random housing of animalsEvidence of caregivers blinded to interventionEvidence of random selection for assessmentEvidence of assessor blindedExplanation of missing animal dataFree of selective reporting based on methods/resultsFree of other high bias risk
Bárcia et al., 2017UnclearYes (Low Risk)UnclearYes (Low Risk)No (High Risk)UnclearNo (High Risk)Yes (Low Risk)Yes (Low Risk)No (High Risk)
Bharti et al., 2020UnclearUnclearUnclearYes (Low Risk)UnclearUnclearUnclearUnclearYes (Low Risk)Yes (Low Risk)
Cruz et al., 2015UnclearYes (Low Risk)UnclearYes (Low Risk)UnclearUnclearYes (Low Risk)UnclearYes (Low Risk)Yes (Low Risk)
Curley et al., 2017UnclearYes (Low Risk)UnclearUnclearUnclearUnclearYes (Low Risk)UnclearYes (Low Risk)Yes (Low Risk)
Devaney et al., 2015UnclearYes (Low Risk)UnclearUnclearUnclearUnclearNo (High Risk)Yes (Low Risk)Yes (Low Risk)Yes (Low Risk)
Gramlich et al., 2016No (High Risk)Yes (Low Risk)UnclearUnclearUnclearUnclearYes (Low Risk)UnclearYes (Low Risk)No (High Risk)
Horie et al., 2020aUnclearUnclearUnclearUnclearUnclearUnclearYes (Low Risk)UnclearYes (Low Risk)Yes (Low Risk)
Horie et al., 2020aUnclearUnclearUnclearUnclearUnclearUnclearUnclearYes (Low Risk)Yes (Low Risk)Yes (Low Risk)
Khan et al., 2019UnclearYes (Low Risk)UnclearUnclearYes (Low Risk)UnclearYes (Low Risk)Yes (Low Risk)Yes (Low Risk)Yes (Low Risk)
Lohan et al., 2018No (High Risk)UnclearUnclearUnclearUnclearUnclearUnclearUnclearNo (High Risk)Yes (Low Risk)
Perlee et al., 2019No (High Risk)UnclearUnclearYes (Low Risk)UnclearUnclearYes (Low Risk)UnclearYes (Low Risk)Yes (Low Risk)
Rogulska et al., 2019UnclearYes (Low Risk)UnclearYes (Low Risk)UnclearUnclearYes (Low Risk)UnclearYes (Low Risk)No (High Risk)
Salmenkari et al., 2019No (High Risk)Yes (Low Risk)UnclearYes (Low Risk)UnclearUnclearYes (Low Risk)Yes (Low Risk)Yes (Low Risk)Yes (Low Risk)
Somal et al., 2017No (High Risk)UnclearUnclearYes (Low Risk)UnclearUnclearUnclearUnclearYes (Low Risk)Yes (Low Risk)
Tan et al., 2019Yes (Low Risk)Yes (Low Risk)Yes (Low Risk)UnclearYes (Low Risk)UnclearYes (Low Risk)Yes (Low Risk)Yes (Low Risk)Yes (Low Risk)
Yea et al., 2020UnclearYes (Low Risk)UnclearYes (Low Risk)UnclearUnclearUnclearUnclearYes (Low Risk)Yes (Low Risk)
Horiuchi et al., 2021UnclearYes (Low Risk)UnclearYes (Low Risk)UnclearUnclearUnclearUnclearYes (Low Risk)Yes (Low Risk)
Horie et al., 2021UnclearYes (Low Risk)Yes (Low Risk)Yes (Low Risk)UnclearUnclearYes (Low Risk)UnclearYes (Low Risk)Yes (Low Risk)
Table 4
All in vivo outcomes where freshly cultured vs. cryopreserved MSCs have been compared directly are reported.
StudyAnimal ModelOutcomeNumber (n)Type and Source of MSCsDuration of Culture Post-Thaw (hr)Concentration of MSCsPre-Treatment of MSCsNegative Control (NC)Positive Control (PC)p-value for Fresh MSCs vs. controlp-value for Frozen MSCs vs. controlFresh or Frozen MSC more effective?p-value for Fresh vs. Frozen comparison
Acute Lung Injury and Sepsis
Devaney et al., 2015Acute lung injury induced by E. coli pneumonia in ratsArterial oxygenation10Human Bone Marrow01×10^7 hMSCs/kgN/AN/APBS<0.05<0.05↔NS
Lung compliance10Human Bone Marrow01×10^7 hMSCs/kgN/AN/APBS<0.05<0.05↔NS
BAL protein10Human Bone Marrow01×10^7 hMSCs/kgN/AN/APBS<0.05<0.05↔NS
BAL neutrophils10Human Bone Marrow01×10^7 hMSCs/kgN/AN/APBS<0.05<0.05↔NS
BAL E. coli bacterial load10Human Bone Marrow01×10^7 hMSCs/kgN/AN/APBS<0.05<0.05↔NS
BAL IL-610Human Bone Marrow01×10^7 hMSCs/kgN/AN/APBS<0.05<0.05↔NS
BAL IL-1010Human Bone Marrow01×10^7 hMSCs/kgN/AN/APBS<0.05<0.05↔NS
Cruz et al., 2015Allergic Airways Inflammation induced by Aspergillus hyphal extract (AHE) exposure in mice.Large Airway Resistance10 (Fresh) and 7 (Frozen)Human Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
Large Airway Resistance6Murine Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
Overall Tissue Resistance10 (Fresh) and 7 (Frozen)Human Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
Overall Tissue Resistance6Murine Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
Lung Elastance10 (Fresh) and 7 (Frozen)Human Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
Lung Elastance6Murine Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
Inflammation Score10 (Fresh) and 7 (Frozen)Human Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
Inflammation Score6Murine Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
BALF Total Cell Number10 (Fresh) and 7 (Frozen)Human Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
BALF Total Cell Number6Murine Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
BAL Neutrophils10 (Fresh) and 7 (Frozen)Human Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
BAL Neutrophils6Murine Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
BAL Eosinophils10 (Fresh) and 7 (Frozen)Human Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
BAL Eosinophils6Murine Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
BAL Macrophages10 (Fresh) and 7 (Frozen)Human Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
BAL Macrophages6Murine Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
BAL Lymphocytes10 (Fresh) and 7 (Frozen)Human Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
BAL Lymphocytes6Murine Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05Frozen better<0.05
BAL IL-1a10 (Fresh) and 7 (Frozen)Human Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
BAL IL-1a6Murine Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
BAL IL-310 (Fresh) and 7 (Frozen)Human Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
BAL IL-36Murine Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
BAL IL-410 (Fresh) and 7 (Frozen)Human Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
BAL IL-46Murine Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
BAL IL-510 (Fresh) and 7 (Frozen)Human Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
BAL IL-56Murine Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
BAL IL-610 (Fresh) and 7 (Frozen)Human Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
BAL IL-66Murine Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
BAL IL-1010 (Fresh) and 7 (Frozen)Human Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
BAL IL-106Murine Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
BAL IL-12-p4010 (Fresh) and 7 (Frozen)Human Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
BAL IL-12-p406Murine Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
BAL IL-1310 (Fresh) and 7 (Frozen)Human Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
BAL IL-136Murine Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
BAL IL-1710 (Fresh) and 7 (Frozen)Human Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05Fresh better<0.05
BAL IL-176Murine Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
BAL KC10 (Fresh) and 7 (Frozen)Human Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05Fresh better<0.05
BAL KC6Murine Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05Frozen better<0.05
BAL RANTES10 (Fresh) and 7 (Frozen)Human Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
BAL RANTES6Murine Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
IFN-y10 (Fresh) and 7 (Frozen)Human Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
IFN-y6Murine Bone Marrow01 × 10^6 viable MSC cellsFrozen MSCs washed 3 times prior to useNaïve (PBS model)AHE +PBS,
Human Lung Fibroblasts		<0.05<0.05↔NS
Curley et al., 2017Acute respiratory distress syndrome by intratracheal instillation of E. coli in rats.Arterial Oxygenation (FiO2=0.3)8–10Human Umbilical Cord (Frozen) and Bone marrow (Fresh) MSCsNR1×10^7 MSCs/kgN/ASham model +PBSE. coli+PBS<0.05<0.05↔NS
Arterial Oxygenation (FiO2=1)8–10Human Umbilical Cord (Frozen) and Bone marrow (Fresh) MSCsNR1×10^7 MSCs/kgN/ASham model +PBSE. coli+PBS<0.05<0.05↔NS
Lung Compliance8–10Human Umbilical Cord (Frozen) and Bone marrow (Fresh) MSCsNR1×10^7 MSCs/kgN/ASham model +PBSE. coli+PBS<0.05<0.05↔NS
Wet:Dry Lung Ratio8–10Human Umbilical Cord (Frozen) and Bone marrow (Fresh) MSCsNR1×10^7 MSCs/kgN/ASham model +PBSE. coli+PBS<0.05<0.05↔NS
BAL Neutrophils8–10Human Umbilical Cord (Frozen) and Bone marrow (Fresh) MSCsNR1×10^7 MSCs/kgN/ASham model +PBSE. coli+PBS<0.05<0.05↔NS
BAL Bacteria8–10Human Umbilical Cord (Frozen) and Bone marrow (Fresh) MSCsNR1×10^7 MSCs/kgN/ASham model +PBSE. coli+PBS<0.05<0.05↔NS
Bárcia et al., 20171) Chronic adjuvant-induced arthritis (AIA) model
2) Hindlimb ischemia model in mice		Arthritis Index6Human Umbilical Cord MSCs01.7×10^6 MSCsFresh MSCs were cryopreserved and then cultured for up to 5 daysSham model +PBSN/AP<0.0001P<0.0001↔NS
Left Paw Volume6Human Umbilical Cord MSCs01.7×10^6 MSCsFresh MSCs were cryopreserved and then cultured for up to 5 daysSham model +PBSN/AP<0.0001P<0.0001↔NS
Right Paw Volume6Human Umbilical Cord MSCs01.7×10^6 MSCsFresh MSCs were cryopreserved and then cultured for up to 5 daysSham model +PBSN/AP<0.0001P<0.0001↔NS
Weight6Human Umbilical Cord MSCs01.7×10^6 MSCsFresh MSCs were cryopreserved and then cultured for up to 5 daysSham model +PBSN/AP<0.0001P<0.0001↔NS
Blood Flow Ratio in Hindlimb D012Human Umbilical Cord MSCs02×10^5 MSCsFresh MSCs were cryopreserved and then cultured for up to 5 daysN/APBSNSNS↔NS
Blood Flow Ratio in Hindlimb D712Human Umbilical Cord MSCs02×10^5 MSCsFresh MSCs were cryopreserved and then cultured for up to 5 daysN/APBSP=0.008P=0.019↔NS
Blood Flow Ratio in Hindlimb D1412Human Umbilical Cord MSCs02×10^5 MSCsFresh MSCs were cryopreserved and then cultured for up to 5 daysN/APBSP=0.012P=0.031↔NS
Blood Flow Ratio in Hindlimb D2112Human Umbilical Cord MSCs02×10^5 MSCsFresh MSCs were cryopreserved and then cultured for up to 5 daysN/APBSP=0.004P=0.002↔NS
Salmenkari et al., 2019Acute phase and Regenerative Phase of Colitis model in miceMacroscopic Score9Human Bone MarrowNR0.5 x
10^6 MSCs		N/ASham model with PBSColitis +VehiclePC: NSPC: NS↔NS
Colon Weight (% change)9Human Bone MarrowNR0.5 x
10^6 MSCs		N/ASham model with PBSColitis +VehiclePC: NS
NC = P=0.001		PC: NS
NC: P=0.001		↔NS
Colon Length9Human Bone MarrowNR0.5 x
10^6 MSCs		N/ASham model with PBSColitis +VehiclePC: NS
NC = P=0.018		PC: NS
NC: P=0.014		↔NS
Histopathology Scpre9Human Bone MarrowNR0.5 x
10^6 MSCs		N/ASham model with PBSColitis +VehiclePC: NS
NC = P=0.004		PC: NS
NC: P=0.001		↔NS
Regeneration9Human Bone MarrowNR0.5 x
10^6 MSCs		N/ASham model with PBSColitis +VehiclePC: NSPC: NS↔NS
IL-1b in colon tissue homogenates9Human Bone MarrowNR0.5 x
10^6 MSCs		N/ASham model with PBSColitis +VehiclePC: NSPC: NS↔NS
TNFa in colon tissue homogenates9Human Bone MarrowNR0.5 x
10^6 MSCs		N/ASham model with PBSColitis +VehiclePC: NSPC: NS↔NS
IL-1b mRNA in colon9Human Bone MarrowNR0.5 x
10^6 MSCs		N/ASham model with PBSColitis +VehiclePC: NSPC: NS↔NS
Corticosterone in colon tissue homogenates9Human Bone MarrowNR0.5 x
10^6 MSCs		N/ASham model with PBSColitis +VehiclePC: NSPC: NS↔NS
Tissue ACE levels9Human Bone MarrowNR0.5 x
10^6 MSCs		N/ASham model with PBSColitis +VehiclePC: NSPC: P<0.05↔NS
Atgr1a mRNA expression9Human Bone MarrowNR0.5 x
10^6 MSCs		N/ASham model with PBSColitis +VehiclePC: NSPC: NS↔NS
ACE shedding9Human Bone MarrowNR0.5 x
10^6 MSCs		N/ASham model with PBSColitis +VehiclePC: NSPC: P<0.001↔NS
Somal et al., 2017Wound Healing of surgical dorsal limb wound in ratsWound Area D03Caprine Amniotic FluidNR1 × 10^6 MSC cellsN/AN/APBSNSNS↔NS
Wound Area D73Caprine Amniotic FluidNR1 × 10^6 MSC cellsN/AN/APBSP<0.05P<0.05↔NS
Wound Area D143Caprine Amniotic FluidNR1 × 10^6 MSC cellsN/AN/APBSNSNS↔NS
Wound Area D213Caprine Amniotic FluidNR1 × 10^6 MSC cellsN/AN/APBSNSNS↔NS
Wound Area D283Caprine Amniotic FluidNR1 × 10^6 MSC cellsN/AN/APBSNSNS↔NS
% Wound Contraction D73Caprine Amniotic FluidNR1 × 10^6 MSC cellsN/AN/APBSP<0.05NS↔NS
% Wound Contraction D143Caprine Amniotic FluidNR1 × 10^6 MSC cellsN/AN/APBSNSNS↔NS
% Wound Contraction D213Caprine Amniotic FluidNR1 × 10^6 MSC cellsN/AN/APBSNSNS↔NS
% Wound Contraction D283Caprine Amniotic FluidNR1 × 10^6 MSC cellsN/AN/APBSNSNS↔NS
Epithelization3Caprine Amniotic FluidNR1 × 10^6 MSC cellsN/AN/APBSP<0.05P<0.05↔NS
Neovascularization3Caprine Amniotic FluidNR1 × 10^6 MSC cellsN/AN/APBSP<0.05P<0.05↔NS
Collagen Thickness3Caprine Amniotic FluidNR1 × 10^6 MSC cellsN/AN/APBSP<0.05P<0.05↔NS
Collagen Density3Caprine Amniotic FluidNR1 × 10^6 MSC cellsN/AN/APBSP<0.05P<0.05↔NS
Wound Area D03Caprine Amniotic SacNR1 × 10^6 MSC cellsN/AN/APBSNSNS↔NS
Wound Area D73Caprine Amniotic SacNR1 × 10^6 MSC cellsN/AN/APBSNSNS↔NS
Wound Area D143Caprine Amniotic SacNR1 × 10^6 MSC cellsN/AN/APBSNSNS↔NS
Wound Area D213Caprine Amniotic SacNR1 × 10^6 MSC cellsN/AN/APBSNSNS↔NS
Wound Area D283Caprine Amniotic SacNR1 × 10^6 MSC cellsN/AN/APBSNSNS↔NS
% Wound Contraction D73Caprine Amniotic SacNR1 × 10^6 MSC cellsN/AN/APBSNSNS↔NS
% Wound Contraction D143Caprine Amniotic SacNR1 × 10^6 MSC cellsN/AN/APBSNSNS↔NS
% Wound Contraction D213Caprine Amniotic SacNR1 × 10^6 MSC cellsN/AN/APBSNSNS↔NS
% Wound Contraction D283Caprine Amniotic SacNR1 × 10^6 MSC cellsN/AN/APBSNSNS↔NS
Epithelization3Caprine Amniotic SacNR1 × 10^6 MSC cellsN/AN/APBSP<0.05P<0.05↔NS
Neovascularization3Caprine Amniotic SacNR1 × 10^6 MSC cellsN/AN/APBSP<0.05P<0.05↔NS
Collagen Thickness3Caprine Amniotic SacNR1 × 10^6 MSC cellsN/AN/APBSNSP<0.05↔NS
Collagen Density3Caprine Amniotic SacNR1 × 10^6 MSC cellsN/AN/APBSP<0.05P<0.05↔NS
Wound Area D03Caprine Wharton’s JellyNR1 × 10^6 MSC cellsN/AN/APBSNSNS↔NS
Wound Area D73Caprine Wharton’s JellyNR1 × 10^6 MSC cellsN/AN/APBSP<0.05NS↔NS
Wound Area D143Caprine Wharton’s JellyNR1 × 10^6 MSC cellsN/AN/APBSNSNS↔NS
Wound Area D213Caprine Wharton’s JellyNR1 × 10^6 MSC cellsN/AN/APBSNSNS↔NS
Wound Area D283Caprine Wharton’s JellyNR1 × 10^6 MSC cellsN/AN/APBSNSNS↔NS
% Wound Contraction D73Caprine Wharton’s JellyNR1 × 10^6 MSC cellsN/AN/APBSP<0.05NS↔NS
% Wound Contraction D143Caprine Wharton’s JellyNR1 × 10^6 MSC cellsN/AN/APBSNSNS↔NS
% Wound Contraction D213Caprine Wharton’s JellyNR1 × 10^6 MSC cellsN/AN/APBSNSNS↔NS
% Wound Contraction D283Caprine Wharton’s JellyNR1 × 10^6 MSC cellsN/AN/APBSNSNS↔NS
Epithelization3Caprine Wharton’s JellyNR1 × 10^6 MSC cellsN/AN/APBSP<0.05P<0.05↔NS
Neovascularization3Caprine Wharton’s JellyNR1 × 10^6 MSC cellsN/AN/APBSP<0.05P<0.05↔NS
Collagen Thickness3Caprine Wharton’s JellyNR1 × 10^6 MSC cellsN/AN/APBSP<0.05P<0.05↔NS
Collagen Density3Caprine Wharton’s JellyNR1 × 10^6 MSC cellsN/AN/APBSP<0.05P<0.05↔NS
Wound Area D03Caprine Cord BloodNR1 × 10^6 MSC cellsN/AN/APBSNSNS↔NS
Wound Area D73Caprine Cord BloodNR1 × 10^6 MSC cellsN/AN/APBSP<0.05NS↔NS
Wound Area D143Caprine Cord BloodNR1 × 10^6 MSC cellsN/AN/APBSNSNS↔NS
Wound Area D213Caprine Cord BloodNR1 × 10^6 MSC cellsN/AN/APBSNSNS↔NS
Wound Area D283Caprine Cord BloodNR1 × 10^6 MSC cellsN/AN/APBSNSNS↔NS
% Wound Contraction D73Caprine Cord BloodNR1 × 10^6 MSC cellsN/AN/APBSP<0.05NS↔NS
% Wound Contraction D143Caprine Cord BloodNR1 × 10^6 MSC cellsN/AN/APBSNSNS↔NS
% Wound Contraction D213Caprine Cord BloodNR1 × 10^6 MSC cellsN/AN/APBSNSNS↔NS
% Wound Contraction D283Caprine Cord BloodNR1 × 10^6 MSC cellsN/AN/APBSNSNS↔NS
Epithelization3Caprine Cord BloodNR1 × 10^6 MSC cellsN/AN/APBSP<0.05P<0.05↔NS
Neovascularization3Caprine Cord BloodNR1 × 10^6 MSC cellsN/AN/APBSP<0.05P<0.05↔NS
Collagen Thickness3Caprine Cord BloodNR1 × 10^6 MSC cellsN/AN/APBSNSNS↔NS
Collagen Density3Caprine Cord BloodNR1 × 10^6 MSC cellsN/AN/APBSP<0.05NSFrozen betterP<0.05
Lohan et al., 2018Corneal Transplantation in ratsOpacity Score, measured from day 5 post-implantation to day 30Fresh = 13,
Frozen = 10		Rat Bone Marrow01×10^6 MSCFrozen MSCs pre-treated with allogenic splenocytes, and co-intervention with MMF.
No MMF for Fresh MSCs.		N/ATransplantation +No treatmentNSNS↔NR
Neovascularization Score, measured from day 5 post-implantation to day 30Fresh = 13,
Frozen = 10		Rat Bone Marrow01×10^6 MSCFrozen MSCs pre-treated with allogenic splenocytes, and co-intervention with MMF.
No MMF for Fresh MSCs.		N/ATransplantation +No treatmentP<0.001NS↔NR
Gramlich et al., 2016Retinal ischemia/reperfusion model in miceRetinal ganglion cells/mm^2Fresh = 10,
Frozen = 8		Human MSCs<1 hr3×10^4 MSCN/ASham modelPBSP=0.019P=0.024↔NS
Perlee et al., 2019Pneumosepsis Caused by Klebsiella
pneumoniae		Lung Bacterial Load at 16 hours8Human Adipose Tissue01×10^6 ASCsMSCs infused at 1 or 6 hours
after infection.		N/APBSNSP<0.001↔NS
Lung Bacterial Load at 48 hours8Human Adipose Tissue01×10^6 ASCsMSCs infused at 1 or 6 hours
after infection.		N/APBSP<0.0001P<0.001↔NS
Blood Bacterial Load at 16 hours8Human Adipose Tissue01×10^6 ASCsMSCs infused at 1 or 6 hours
after infection.		N/APBSNSNS↔NS
Blood Bacterial Load at 48 hours8Human Adipose Tissue01×10^6 ASCsMSCs infused at 1 or 6 hours
after infection.		N/APBSP<0.001P<0.001↔NS
Liver Bacterial Load at 16 hours8Human Adipose Tissue01×10^6 ASCsMSCs infused at 1 or 6 hours
after infection.		N/APBSNSNS↔NS
Liver Bacterial Load at 48 hours8Human Adipose Tissue01×10^6 ASCsMSCs infused at 1 or 6 hours
after infection.		N/APBSP<0.0001P<0.001↔NS
Spleen Bacterial Load at 16 hours8Human Adipose Tissue01×10^6 ASCsMSCs infused at 1 or 6 hours
after infection.		N/APBSNSNS↔NS
Spleen Bacterial Load at 48 hours8Human Adipose Tissue01×10^6 ASCsMSCs infused at 1 or 6 hours
after infection.		N/APBSP<0.001P<0.01↔NS
Lung TNFa at 16 hours8Human Adipose Tissue01×10^6 ASCsMSCs infused at 1 or 6 hours
after infection.		N/APBSP<0.0001P<0.05↔NS
Lung TNFa at 48 hours8Human Adipose Tissue01×10^6 ASCsMSCs infused at 1 or 6 hours
after infection.		N/APBSP<0.001P<0.05↔NS
Lung IL-1b at 16 hours8Human Adipose Tissue01×10^6 ASCsMSCs infused at 1 or 6 hours
after infection.		N/APBSP<0.05P<0.01↔NS
Lung IL-1b at 48 hours8Human Adipose Tissue01×10^6 ASCsMSCs infused at 1 or 6 hours
after infection.		N/APBSP<0.001P<0.05↔NS
Lung IL-6 at 16 hours8Human Adipose Tissue01×10^6 ASCsMSCs infused at 1 or 6 hours
after infection.		N/APBSP<0.05P<0.01↔NS
Lung IL-6 at 48 hours8Human Adipose Tissue01×10^6 ASCsMSCs infused at 1 or 6 hours
after infection.		N/APBSP<0.01NS↔NS
MIP-2 at 16 hours8Human Adipose Tissue01×10^6 ASCsMSCs infused at 1 or 6 hours
after infection.		N/APBSP<0.05P<0.01↔NS
MIP-2 at 48 hours8Human Adipose Tissue01×10^6 ASCsMSCs infused at 1 or 6 hours
after infection.		N/APBSP<0.001P<0.05↔NS
Horie et al., 2020aE. coli-induced lung injury.Arterial Oxygenation8Human Umbilical Cord01 ×
10^7 MSCs/kg		Isolated CD362+MSCs for useN/APBSP<0.05P<0.05↔NS
Lung Wet:Dry Ratio8Human Umbilical Cord01 ×
10^7 MSCs/kg		Isolated CD362+MSCs for useN/APBSNSNS↔NS
Lung Compliance8Human Umbilical Cord01 ×
10^7 MSCs/kg		Isolated CD362+MSCs for useN/APBSP<0.05NS↔NS
BAL E. coli Counts8Human Umbilical Cord01 ×
10^7 MSCs/kg		Isolated CD362+MSCs for useN/APBSP<0.05P<0.05↔NS
BAL WCC levels8Human Umbilical Cord01 ×
10^7 MSCs/kg		Isolated CD362+MSCs for useN/APBSP<0.05P<0.05↔NS
BAL Neutrophils8Human Umbilical Cord01 ×
10^7 MSCs/kg		Isolated CD362+MSCs for useN/APBSP<0.05P<0.05↔NS
BAL IL-1b8Human Umbilical Cord01 ×
10^7 MSCs/kg		Isolated CD362+MSCs for useN/APBSP<0.05P<0.05↔NS
BAL CINC-18Human Umbilical Cord01 ×
10^7 MSCs/kg		Isolated CD362+MSCs for useN/APBSNSNS↔NS
BAL IL-68Human Umbilical Cord01 ×
10^7 MSCs/kg		Isolated CD362+MSCs for useN/APBSP<0.05P<0.05↔NS
Horie et al., 2020aVentilator-induced Lung InjuryArterial OxygenationFresh, n=7–8; Cryopreserved, n=5–
6		Human Bone MarrowNR1×10^7 MSCs/
kg		Pre-activated MSCs (fresh and frozen were also used)Sham modelPBSP<0.001P<0.001↔NS
Lung ComplianceFresh, n=7–8; Cryopreserved, n=5–
6		Human Bone MarrowNR1×10^7 MSCs/
kg		Pre-activated MSCs (fresh and frozen were also used)Sham modelPBSNSNS↔NS
Lung Wet:Dry RatioFresh, n=7–8; Cryopreserved, n=5–
6		Human Bone MarrowNR1×10^7 MSCs/
kg		Pre-activated MSCs (fresh and frozen were also used)Sham modelPBSP<0.05P<0.05↔NS
BAL ProteinFresh, n=7–8; Cryopreserved, n=5–
6		Human Bone MarrowNR1×10^7 MSCs/
kg		Pre-activated MSCs (fresh and frozen were also used)Sham modelPBSNSNS↔NS
Percentage of Alveolar AirspaceFresh, n=8;
Cryopreserved, n=6		Human Bone MarrowNR1×10^7 MSCs/
kg		Pre-activated MSCs (fresh and frozen were also used)Sham modelPBSP<0.001P<0.001↔NS
BAL NeutrophilsFresh, n=6–8;
Cryopreserved, n=5–6		Human Bone MarrowNR1×10^7 MSCs/
kg		Pre-activated MSCs (fresh and frozen were also used)Sham modelPBSP<0.05P<0.01↔NS
BAL CINC-1Fresh, n=6–8;
Cryopreserved, n=5–6		Human Bone MarrowNR1×10^7 MSCs/
kg		Pre-activated MSCs (fresh and frozen were also used)Sham modelPBSP<0.05P<0.05↔NS
BAL IL-6Fresh, n=6–8;
Cryopreserved, n=5–6		Human Bone MarrowNR1×10^7 MSCs/
kg		Pre-activated MSCs (fresh and frozen were also used)Sham modelPBSP<0.05P<0.001↔NS
BAL IL-10Fresh, n=6–8;
Cryopreserved, n=5–6		Human Bone MarrowNR1×10^7 MSCs/
kg		Pre-activated MSCs (fresh and frozen were also used)Sham modelPBSNSNS↔NS
BAL KGFFresh, n=6–8;
Cryopreserved, n=5–6		Human Bone MarrowNR1×10^7 MSCs/
kg		Pre-activated MSCs (fresh and frozen were also used)Sham modelPBSNSNS↔NS
BAL PGE2Fresh, n=6–8;
Cryopreserved, n=5–6		Human Bone MarrowNR1×10^7 MSCs/
kg		Pre-activated MSCs (fresh and frozen were also used)Sham modelPBSNSNS↔NS
Tan et al., 2019Polymicrobial sepsis induced by cecal-ligation-and-puncture (CLP)%CD11b+/E. coli+cells in Peritoneal FluidFresh, n=12;
Cryopreserved, n=11		Human Bone Marrow02.5×10^5 MSC cellsN/ASham modelPBSP<0.0001P<0.0001↔NS
Peritoneal CFU #Fresh, n=12;
Cryopreserved, n=11		Human Bone Marrow02.5×10^5 MSC cellsN/ASham modelPBSNSNS↔NS
Plasma LactateFresh, n=12;
Cryopreserved, n=11		Human Bone Marrow02.5×10^5 MSC cellsN/ASham modelPBSP<0.05P<0.05↔NS
Plasma CCL5Fresh, n=12;
Cryopreserved, n=11		Human Bone Marrow02.5×10^5 MSC cellsN/ASham modelPBSNSP<0.01↔NS
Plasma JEFresh, n=12;
Cryopreserved, n=11		Human Bone Marrow02.5×10^5 MSC cellsN/ASham modelPBSNSNS↔NS
Plasma KCFresh, n=12;
Cryopreserved, n=11		Human Bone Marrow02.5×10^5 MSC cellsN/ASham modelPBSP<0.05NS↔NS
Plasma LIXFresh, n=12;
Cryopreserved, n=11		Human Bone Marrow02.5×10^5 MSC cellsN/ASham modelPBSNSNS↔NS
Plasma IL-10Fresh, n=12;
Cryopreserved, n=11		Human Bone Marrow02.5×10^5 MSC cellsN/ASham modelPBSNSNS↔NS
Plasma IL-1bFresh, n=12;
Cryopreserved, n=11		Human Bone Marrow02.5×10^5 MSC cellsN/ASham modelPBSNSNS↔NS
Bharti et al., 2020Wound healing model with 2×2 cm^2
full-thickness excision skin wound in guinea pigs		Percent wound contraction D75Dog Bone MarrowNR1×10^6 MSC cellsMSCs attached to polypropylene mesh of 2×2 cm2 sizeN/AAntibiotic only, Mesh only, and MSCs only as control groupsNSNS↔NS
Percent wound contraction D145Dog Bone MarrowNR1×10^6 MSC cellsMSCs attached to polypropylene mesh of 2×2 cm2 sizeN/AAntibiotic only, Mesh only, and MSCs only as control groupsP<0.05P<0.05↔NS
Percent wound contraction D215Dog Bone MarrowNR1×10^6 MSC cellsMSCs attached to polypropylene mesh of 2×2 cm2 sizeN/AAntibiotic only, Mesh only, and MSCs only as control groupsP<0.05P<0.05↔NS
Percent wound contraction D285Dog Bone MarrowNR1×10^6 MSC cellsMSCs attached to polypropylene mesh of 2×2 cm2 sizeN/AAntibiotic only, Mesh only, and MSCs only as control groupsP<0.05P<0.05↔NS
Epithelialization5Dog Bone MarrowNR1×10^6 MSC cellsMSCs attached to polypropylene mesh of 2×2 cm2 sizeN/AAntibiotic only, Mesh only, and MSCs only as control groupsP<0.05P<0.05↔NS
Neovascularization5Dog Bone MarrowNR1×10^6 MSC cellsMSCs attached to polypropylene mesh of 2×2 cm2 sizeN/AAntibiotic only, Mesh only, and MSCs only as control groupsP<0.05P<0.05↔NS
Collagen Density5Dog Bone MarrowNR1×10^6 MSC cellsMSCs attached to polypropylene mesh of 2×2 cm2 sizeN/AAntibiotic only, Mesh only, and MSCs only as control groupsP<0.05P<0.05↔NS
Collagen Thickness5Dog Bone MarrowNR1×10^6 MSC cellsMSCs attached to polypropylene mesh of 2×2 cm2 sizeN/AAntibiotic only, Mesh only, and MSCs only as control groupsP<0.05P<0.05↔NS
Rogulska et al., 2019Wound Healing of
Full-thickness excisional skin wounds in mice		Percent Wound Closure D314Human Adipose Tissue24 hours0.25‐0.3×10^6 cells in 50 μlMSCs placed on 3D gel containing PPP, 0.2 M
sucrose, 1% DMSO		N/ASpontaneous healing, and 3D gel containing PPP, 0.2 M
sucrose, 1% DMSO alone		P<0.05P<0.05↔NS
Percent Wound Closure D714Human Adipose Tissue24 hours0.25‐0.3×10^6 cells in 50 μlMSCs placed on 3D gel containing PPP, 0.2 M
sucrose, 1% DMSO		N/ASpontaneous healing, and 3D gel containing PPP, 0.2 M
sucrose, 1% DMSO alone		P<0.05P<0.05↔NS
Percent Wound Closure D1414Human Adipose Tissue24 hours0.25‐0.3×10^6 cells in 50 μlMSCs placed on 3D gel containing PPP, 0.2 M
sucrose, 1% DMSO		N/ASpontaneous healing, and 3D gel containing PPP, 0.2 M
sucrose, 1% DMSO alone		P<0.05P<0.05↔NS
Percent Wound Closure D2814Human Adipose Tissue24 hours0.25‐0.3×10^6 cells in 50 μlMSCs placed on 3D gel containing PPP, 0.2 M
sucrose, 1% DMSO		N/ASpontaneous healing, and 3D gel containing PPP, 0.2 M
sucrose, 1% DMSO alone		P<0.05P<0.05↔NS
Khan et al., 2019Acute Spinal Cord Injury in dogsMotor activity of hind limbs
assessed by using the canine Basso Beattie Bresnahan (cBBB)
score at Week 1		4Dog Adipose Tissue01×10^7 MSC cellsLentivirus Mediated HO-1 Gene Insertion into Ad-
MSCs.		N/AFresh MSCs expressing GFP only.NSNS↔NS
cBBB score at Week 24Dog Adipose Tissue01×10^7 MSC cellsLentivirus Mediated HO-1 Gene Insertion into Ad-
MSCs.		N/AFresh MSCs expressing GFP only.NSNS↔NS
cBBB score at Week 34Dog Adipose Tissue01×10^7 MSC cellsLentivirus Mediated HO-1 Gene Insertion into Ad-
MSCs.		N/AFresh MSCs expressing GFP only.NSNS↔NS
cBBB score at Week 44Dog Adipose Tissue01×10^7 MSC cellsLentivirus Mediated HO-1 Gene Insertion into Ad-
MSCs.		N/AFresh MSCs expressing GFP only.P<0.05NS↔NS
% age of gross lesion area4Dog Adipose Tissue01×10^7 MSC cellsLentivirus Mediated HO-1 Gene Insertion into Ad-
MSCs.		N/AFresh MSCs expressing GFP only.NSNS↔NS
Fibrotic areas relative to normal4Dog Adipose Tissue01×10^7 MSC cellsLentivirus Mediated HO-1 Gene Insertion into Ad-
MSCs.		Normal (no SCI)Fresh MSCs expressing GFP only.P<0.05NS↔NS
Myelinated areas relative to normal4Dog Adipose Tissue01×10^7 MSC cellsLentivirus Mediated HO-1 Gene Insertion into Ad-
MSCs.		Normal (no SCI)Fresh MSCs expressing GFP only.P<0.05NS↔NS
Yea et al., 2020Wound healing in ratsTotal macroscopic score at 2 weeks4Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsP=0.001P=0.04↔NS
Total macroscopic score at 4 weeks4Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsP=0.001P<0.05↔NS
Total degeneration score at 2 weeks4Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsP<0.001P<0.001↔NS
Total degeneration score at 4 weeks4Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsP<0.05P<0.05↔NS
Fibre structure at 2 weeks4Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsNSNS↔NS
Fibre structure at 4 weeks4Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsP<0.05P<0.05↔NS
Fibre arrangement at 2 weeks4Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsNSNS↔NS
Fibre arrangement at 4 weeks4Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsP<0.05P<0.05↔NS
Rounding of nuclei at 2 weeks4Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsNSNS↔NS
Rounding of nuclei at 4 weeks4Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsP<0.05P<0.05↔NS
Variations in cellularity at 2 weeks4Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsNSNS↔NS
Variations in cellularity at 4 weeks4Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsP<0.05P<0.05↔NS
Decreased stainability at 2 weeks4Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsNSNS↔NS
Decreased stainability at 4 weeks4Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsP<0.05P<0.05↔NS
Hyalinization at 2 weeks4Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsNSNS↔NS
Hyalinization at 4 weeks4Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsP<0.05P<0.05↔NS
Inflammation at 2 weeks4Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsNSNS↔NS
Inflammation at 4 weeks4Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsP<0.05P<0.05↔NS
Fibroblast density at 2 weeks4Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsNSNS↔NS
Fibroblast density at 4 weeks4Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsP<0.05P<0.05↔NS
Nuclear aspect ratio at 2 weeks4Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsNSNS↔NS
Nuclear aspect ration at 4 weeks4Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsP<0.05P<0.05↔NS
Nuclear orientation at 2 weeks4Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsP<0.05P<0.05↔NS
Nuclear orientation at 4 weeks4Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsP<0.05P<0.05↔NS
Collagen organization at 2 weeks4Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsP<0.05P<0.05↔NS
Collagen organization at 4 weeks4Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsP<0.05P<0.05↔NS
Collagen fibre coherence at 2 weeks4Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsNSNS↔NS
Collagen fibre coherence at 4 weeks4Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsP<0.05P<0.05↔NS
GAG-rich area at 2 weeks4Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsP<0.05P<0.05↔NS
GAG-rich area at 4 weeks4Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsP<0.05P<0.05↔NS
Ultimate failure load at 2 weeks8Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsP<0.05P<0.05↔NS
Ultimate failure load at 4 weeks8Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsP<0.05P<0.05↔NS
Tendon stiffness at 2 weeks8Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsP<0.05P<0.05↔NS
Tendon stiffness at 4 weeks8Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsNSNS↔NS
Ultimate stress at 2 weeks8Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsP<0.05P<0.05↔NS
Ultimate stress at 4 weeks8Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsP<0.05P<0.05↔NS
Cross-sectional area at 2 weeks8Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsP<0.05P<0.05↔NS
Cross-sectional area at 4 weeks8Human Umbilical CordNR1×10^6 MSC cellsN/ACryoprotectant and PBSFresh-MSCsP<0.05P<0.05↔NS
Horiuchi et al., 2021Osteoarthritis model in ratsBioluminescence9Rat synovial MSCsNR1×10^6 MSC cellsN/APBSFresh-MSCsNRNR↔NS
Tibia gross finding score9Rat synovial MSCsNR1×10^6 MSC cellsN/APBSFresh-MSCsP<0.05P<0.05↔NS
Femur gross finding score9Rat synovial MSCsNR1×10^6 MSC cellsN/APBSFresh-MSCsP<0.05P<0.05↔NS
Tibia OARSI score6Rat synovial MSCsNR1×10^6 MSC cellsN/APBSFresh-MSCsP<0.05P<0.05↔NS
Femur OARSI score6Rat synovial MSCsNR1×10^6 MSC cellsN/APBSFresh-MSCsNSNS↔NS
Horie et al., 2021Ventilator-Induced Lung Injury (VILI) model in ratsArterial oxygenation7Human Umbilical Cord MSCsNR1 × 10^7 MSCs/kgN/APBSFresh MSCsP<0.001P<0.001↔NS
Static Lung Compliance7Human Umbilical Cord MSCsNR1 × 10^7 MSCs/kgN/APBSFresh MSCsP<0.01P<0.01↔NS
Wet:Dry Ratio7Human Umbilical Cord MSCsNR1 × 10^7 MSCs/kgN/APBSFresh MSCsP<0.05P<0.05↔NS
BAL Protein7Human Umbilical Cord MSCsNR1 × 10^7 MSCs/kgN/APBSFresh MSCsP<0.01P<0.01↔NS
BAL Cell count7Human Umbilical Cord MSCsNR1 × 10^7 MSCs/kgN/APBSFresh MSCsP<0.01P<0.01↔NS
BAL Neutrophil count7Human Umbilical Cord MSCsNR1 × 10^7 MSCs/kgN/APBSFresh MSCsP<0.05P<0.05↔NS
BAL IL-6 level7Human Umbilical Cord MSCsNR1 × 10^7 MSCs/kgN/APBSFresh MSCsNSP<0.05Frozen betterP<0.05
BAL IL-1 level7Human Umbilical Cord MSCsNR1 × 10^7 MSCs/kgN/APBSFresh MSCsP<0.05P<0.05↔NS
% Airspace4Human Umbilical Cord MSCsNR1 × 10^7 MSCs/kgN/APBSFresh MSCsP<0.001P<0.001↔NS

  1. ↔ indicates no statistically significant difference of Freshly-cultured and Cryopreserved MSCs.

  2. NS indicates Not Significant- statistical analysis from individual studies did not yield significant difference between Freshly-cultured and Cryopreserved MSCs. NR = Not reported.

  3. If direct comparison of Freshly-cultured vs. Cryopreserved MSC was not presented in the same graph by a study, the results and discussion sections of that study were used to judge efficacy of Freshly-cultured vs. Cryopreserved MSCs for the table above.

Table 5
Summary of similar in-vivo outcomes reported across studies.
Outcome MeasureStudyUnit of MeasurementNumber of samples (n)Fresh MSC MeanFresh MSC Std DevFrozen MSC MeanFrozen MSC Std Dev
Arterial Oxygenation0.128Curley et al., 2017mmHg8 to 10217.7777.93242.7584.14
Devaney et al., 2015mmHg10265.567.86247.6468.232
Horie et al., 2020ammHg873.08411.52669.1489.222
Horie et al., 2021kPa716.520.8516.861.10
Lung ComplianceCurley et al., 2017mL/mmHg8 to 100.8620.0820.8180.098
Devaney et al., 2015mL/mmHg120.822640.1320.7650.128
Horie et al., 2020amL/mmHg80.559390.0890.4510.531
Horie et al., 2021mL/cmH2O70.3630.060.3580.08
Wet:Dry Lung RatioCurley et al., 2017Ratio8 to 104.727790.1884.770.157
Horie et al., 2020aRatio84.76430.0744.940.294
Horie et al., 2021Ratio75.210.365.320.42
BAL IL-6 levelsDevaney et al., 2015pg/ml12348.93207.5363.22142.5
Horie et al., 2020apg/ml8224.67119.86181.51126.72
Horie et al., 2021pg/ml7252.3961.64207.7653.66
% of Wound Contraction on D7Somal et al., 2017Percentage360.07616.6755.67912.755
Bharti et al., 2020Percentage516.1041.06214.5212.123
Rogulska et al., 2019Percentage1451.4025.74152.0694.94
% of Wound Contraction on D14Somal et al., 2017Percentage396.3740.8589.9375.103
Bharti et al., 2020Percentage567.3631.6971.5372.123
Rogulska et al., 2019Percentage1499.0652.899.8662.804
% of Wound Contraction on D21Somal et al., 2017Percentage399.850.68198.5152.89
Bharti et al., 2020Percentage584.1411.9389.4571.769
% of Wound Contraction on D28Somal et al., 2017Percentage3100.433100.2880.681
Bharti et al., 2020Percentage599.5830.88599.4150.885
Table 6
In vitro outcomes where freshly cultured vs. cryopreserved MSCs were compared directly.
StudyOutcomeAssay UsedNumber (n)Type and Source of MSCsTime of cell preparation without MSC (hr)Time of outcome measurement from MSC intervention (hr)Concentration of MSCsPre-Treatment of MSCsNegative Control (NC)Positive Control (PC)p-value for Fresh MSCs vs. controlp-value for Frozen MSCs vs. controlFresh or Frozen MSC more effective?p-value for Fresh vs. Frozen comparison
Bárcia et al., 2017ViabilityTrypan BlueFresh/Cultured (12); cryo <1 yr(12); cryo >3 yrs (5)Human Umbilical Cord MSCsN/A0NRFresh/Cultured MSCs were cryopreserved and then cultured for up to 5 daysN/AN/AN/AN/A↔NS
ApoptosisAnnexin V (and flow cytometry)N/AHuman Umbilical Cord MSCsN/A2NRFresh/Cultured MSCs were cryopreserved and then cultured for up to 5 daysN/ACultured cells incubated with H2O2 (2 mmol/L) for 2 hrNRNR↔NS
Angiogenesis: Number of master junctions (branching points)Matrigel/Human umbilical vein endothelial cell (HUVEC) tube formation assay2Human Umbilical Cord MSCs1161 × 106 cellsFresh/Cultured MSCs were cryopreserved and then cultured for up to 5 days; fresh and cryo co-cultured in basal mediaN/AHUVEC in Basal Media and HUVECs in Basal media with VEGF (100 ng/mL)NRNR↔NS
Angiogenesis: segment/tube lengthMatrigel/Human umbilical vein endothelial cell (HUVEC) tube formation assay2Human Umbilical Cord MSCs1161 × 106 cellsFresh/Cultured MSCs were cryopreserved and then cultured for up to 5 days; fresh and cryo co-cultured in basal mediaN/AHUVEC in Basal Media and HUVECs in Basal media with VEGF (100 ng/mL)NRNR↔NS
Angiogenesis:total mesh areaMatrigel/Human umbilical vein endothelial cell (HUVEC) tube formation assay2Human Umbilical Cord MSCs1161 × 106 cellsFresh/Cultured MSCs were cryopreserved and then cultured for up to 5 days; fresh and cryo co-cultured in basal mediaN/AHUVEC in Basal Media and HUVECs in Basal media with VEGF (100 ng/mL)NRNR↔NS
Gramlich et al., 2016ViabilityTUNEL staining via Apo-Direct Apoptosis Detection Kit5Human MSCsN/A2430,000 MSCsBoth fresh and frozen cells were washed twice, resuspended in PBS and analyzed immediately or after 1 hr storage on wet iceN/AN/AN/AN/AFresh betterP<0.001
ViabilityTUNEL staining via Apo-Direct Apoptosis Detection Kit5Human MSCsN/A4830,000 MSCsBoth fresh and frozen cells were washed twice, resuspended in PBS and analyzed immediately or after 1 hr storage on wet iceN/AN/AN/AN/AFresh betterP<0.001
ViabilityTUNEL staining via Apo-Direct Apoptosis Detection Kit5Human MSCsN/A7230,000 MSCsBoth fresh and frozen cells were washed twice, resuspended in PBS and analyzed immediately or after 1 hr storage on wet iceN/AN/AN/AN/AFresh betterP=0.002
Metabolic Activity (measured by XXT)XTT Assay6Human MSCsN/A2415,000 MSCsN/AN/AN/AN/AN/A↔NS
P=0.352
Metabolic Activity (measured by XXT)XTT Assay6Human MSCsN/A4815,000 MSCsN/AN/AN/AN/AN/A↔NS
P=0.312
Metabolic Activity (measured by XXT)XTT Assay6Human MSCsN/A7215,000MSCsN/AN/AN/AN/AN/A↔NS
P=0.971
IDO activity: unstimulated MSCConcentration of kynurenine in conditioned media6Human MSCN/A48NRN/AN/AN/AN/AN/A↔NS
P=0.998
IDO activity:MSC exposed to IFN-yConcentration of kynurenine in conditioned media6Human MSCN/A48NRN/AN/AN/AN/AN/A↔NS
P=0.099
IDO activity: MSC exposed to IFN-y+TNF aConcentration of kynurenine in conditioned media6Human MSCN/A48NRN/AN/AN/AN/AN/A↔NS
P=0.951
GDF-15: unstimulatedHuman Growth Factor Array Q14Human MSCN/A48200,000 MSCsN/AN/AMedia ControlN/AN/AFrozen betterP=0.01
GDF-15: stimulated with IFN-y/TNF-aHuman Growth Factor Array Q14Human MSCN/A48200,000 MSCsN/AN/AMedia ControlN/AN/A↔NS
P=0.99
IGFBP-2: unstimulatedHuman Growth Factor Array Q14Human MSCN/A48200,000 MSCsN/AN/AMedia ControlN/AN/A↔NS
P=0.32
IGFBP-2: stimulated with IFN-y/TNF-aHuman Growth Factor Array Q14Human MSCN/A48200,000 MSCsN/AN/AMedia ControlN/AN/A↔NS
P=0.68
IGFBP-3: unstimulatedHuman Growth Factor Array Q14Human MSCN/A48200,000 MSCsN/AN/AMedia ControlN/AN/A↔NS
P=0.47
IGFBP-3: stimulated with IFN-y/TNF-aHuman Growth Factor Array Q14Human MSCN/A48200,000 MSCsN/AN/AMedia ControlN/AN/A↔NS
P=0.75
IGFBP-4: unstimulatedHuman Growth Factor Array Q14Human MSCN/A48200,000 MSCsN/AN/AMedia ControlN/AN/A↔NS
P=0.39
IGFBP-6: unstimulatedHuman Growth Factor Array Q14Human MSCN/A48200,000 MSCsN/AN/AMedia ControlN/AN/A↔NS
P=0.69
IGFBP-6: stimulated with IFN-y/TNF-aHuman Growth Factor Array Q14Human MSCN/A48200,000 MSCsN/AN/AMedia ControlN/AN/AFresh betterP=0.03
Insulin: stimulated with IFN-y/TNF-aHuman Growth Factor Array Q14Human MSCN/A48200,000 MSCsN/AN/AMedia ControlN/AN/A↔NS
P=0.71
OPG: unstimulatedHuman Growth Factor Array Q14Human MSCN/A48200,000 MSCsN/AN/AMedia ControlN/AN/A↔NS
P=0.39
OPG: stimulated with IFN-y/TNF-aHuman Growth Factor Array Q14Human MSCN/A48200,000 MSCsN/AN/AMedia ControlN/AN/A↔NS
P=0.65
PDGF-AA: unstimulatedHuman Growth Factor Array Q14Human MSCN/A48200,000 MSCsN/AN/AMedia ControlN/AN/A↔NS
P=0.43
PDGF-AA: stimulated with IFN-y/TNF-aHuman Growth Factor Array Q14Human MSCN/A48200,000 MSCsN/AN/AMedia ControlN/AN/AFrozen betterP=0.04
PIGF: unstimulatedHuman Growth Factor Array Q14Human MSCN/A48200,000 MSCsN/AN/AMedia ControlN/AN/A↔NS
P=0.83
SCF R: stimulated with IFN-y/TNF-aHuman Growth Factor Array Q14Human MSCN/A48200,000 MSCsN/AN/AMedia ControlN/AN/A↔NS
P=0.06
TGFb1: unstimulatedHuman Growth Factor Array Q14Human MSCN/A48200,000 MSCsN/AN/AMedia ControlN/AN/AN/AN/A
TGFb1: stimulated with IFN-y/TNF-aHuman Growth Factor Array Q14Human MSCN/A48200,000 MSCsN/AN/AMedia ControlN/AN/AFresh betterP=0.05
VEGF: unstimulatedHuman Growth Factor Array Q14Human MSCN/A48200,000 MSCsN/AN/AMedia ControlN/AN/A↔NS
P=0.30
VEGF: stimulated with IFN-y/TNF-aHuman Growth Factor Array Q14Human MSCN/A48200,000 MSCsN/AN/AMedia ControlN/AN/A↔NS
P=0.96
Tan et al., 2019ViabilityTrypan BlueNRHuman BMN/A0NRN/AN/AN/AN/AN/A↔NS
ViabilityTrypan BlueNRHuman BMN/A2NRN/AN/AN/AN/AN/AFresh betterP<0.05
ViabilityTrypan BlueNRHuman BMN/A4NRN/AN/AN/AN/AN/A↔NS
ViabilityTrypan BlueNRHuman BMN/A6NRN/AN/AN/AN/AN/A↔NS
Viability (Viable Cells)Annexin V+Propidium iodide (AV/PI)NRHuman BMN/A0NRN/AN/AN/AN/AN/A↔NS
Viability (Viable Cells)Annexin V+Propidium iodide (AV/PI)NRHuman BMN/A2NRN/AN/AN/AN/AN/A↔NS
Viability (Viable Cells)Annexin V+Propidium iodide (AV/PI)NRHuman BMN/A4NRN/AN/AN/AN/AN/A↔NS
Viability (Viable Cells)Annexin V+Propidium iodide (AV/PI)NRHuman BMN/A6NRN/AN/AN/AN/AN/AFresh betterP<0.05
Viability(Early apoptotic cells)Annexin V+Propidium iodide (AV/PI)NRHuman BMN/A0NRN/AN/AN/AN/AN/A↔NS
Viability(Early apoptotic cells)Annexin V+Propidium iodide (AV/PI)NRHuman BMN/A2NRN/AN/AN/AN/AN/A↔NS
Viability(Early apoptotic cells)Annexin V+Propidium iodide (AV/PI)NRHuman BMN/A4NRN/AN/AN/AN/AN/A↔NS
Viability(Early apoptotic cells)Annexin V+Propidium iodide (AV/PI)NRHuman BMN/A6NRN/AN/AN/AN/AN/AFresh betterP<0.05
Viability (Late apoptotic cells)Annexin V+Propidium iodide (AV/PI)NRHuman BMN/A0NRN/AN/AN/AN/AN/A↔NS
Viability (Late apoptotic cells)Annexin V+Propidium iodide (AV/PI)NRHuman BMN/A2NRN/AN/AN/AN/AN/A↔NS
Viability (Late apoptotic cells)Annexin V+Propidium iodide (AV/PI)NRHuman BMN/A4NRN/AN/AN/AN/AN/AFresh betterP<0.05
Viability (Late apoptotic cells)Annexin V+Propidium iodide (AV/PI)NRHuman BMN/A6NRN/AN/AN/AN/AN/AFresh betterP<0.05
PhagocytosisPBMCs pre-treated with LPS the co-culture with MSC at ratio of 1:5 for 24 hr3–6Human BM MSC: Donor 1N/A24NRN/ANaive PBMCLPS treated PBMCPC: P<0.0001PC: P<0.0001↔NS
PhagocytosisPBMCs pre-treated with LPS the co-culture with MSC at ratio of 1:5 for 24 hr3–6Human BM MSC: Donor 2N/A24NRN/ANaive PBMCLPS treated PBMCNSNS↔NS
PhagocytosisPBMCs pre-treated with LPS the co-culture with MSC at ratio of 1:5 for 24 hr3–6Human BM MSC: Donor 3N/A24NRN/ANaive PBMCLPS treated PBMCPC: P<0.001PC: P<0.001↔NS
PermeabilityEndothelial cell (EC) treated with LPS for 6 hr then co-culture with MSC for 24 hr at ratio of 1:2 followed by adding FITC-dextran to the transwell insertNRHuman BM MSC: Donor 1N/A24NRN/ANon-treated ECLPS treated ECPC: P<0.01PC: P<0.01↔NS
PermeabilityEndothelial cell (EC) treated with LPS for 6 hr then co-culture with MSC for 24 hr at ratio of 1:2 followed by adding FITC-dextran to the transwell insertNRHuman BM MSC: Donor 2N/A24NRN/ANon-treated ECLPS treated ECPC: P<0.01PC: P<0.01↔NS
PermeabilityEndothelial cell (EC) treated with LPS for 6 hr then co-culture with MSC for 24 hr at ratio of 1:2 followed by adding FITC-dextran to the transwell insertNRHuman BM MSC: Donor 3N/A24NRN/ANon-treated ECLPS treated ECPC: P<0.001PC: P<0.001↔NS
Bharti et al., 2020Growth CurveCountess automated cell counterNRCanine BMN/A241 × 104 cells/mlFrozen cells were thawed in distilled water at 36 °C for 45–60 s then enzymatically detached from mesh and added in re-warmed media with 15% FBS and washed twice at 1200 rpm for 5 minN/AN/AN/AN/A↔NS
Growth CurveCountess automated cell counterNRCanine BMN/A481 × 104 cells/mlFrozen cells were thawed in distilled water at 36 °C for 45–60 s then enzymatically detached from mesh and added in re-warmed media with 15% FBS and washed twice at 1200 rpm for 5 minN/AN/AN/AN/A↔NS
Growth CurveCountess automated cell counterNRCanine BMN/A721 × 104 cells/mlFrozen cells were thawed in distilled water at 36 °C for 45–60 s then enzymatically detached from mesh and added in re-warmed media with 15% FBS and washed twice at 1200 rpm for 5 minN/AN/AN/AN/A↔NS
Growth CurveCountess automated cell counterNRCanine BMN/A961 × 104 cells/mlFrozen cells were thawed in distilled water at 36 °C for 45–60 s then enzymatically detached from mesh and added in re-warmed media with 15% FBS and washed twice at 1200 rpm for 5 minN/AN/AN/AN/A↔NS
Growth CurveCountess automated cell counterNRCanine BMN/A1201 × 104 cells/mlFrozen cells were thawed in distilled water at 36 °C for 45–60 s then enzymatically detached from mesh and added in re-warmed media with 15% FBS and washed twice at 1200 rpm for 5 minN/AN/AN/AN/A↔NS
Growth CurveCountess automated cell counterNRCanine BMN/A1441 × 104 cells/mlFrozen cells were thawed in distilled water at 36 °C for 45–60 s then enzymatically detached from mesh and added in re-warmed media with 15% FBS and washed twice at 1200 rpm for 5 minN/AN/AN/AN/A↔NS
Growth CurveCountess automated cell counterNRCanine BMN/A1681 × 104 cells/mlFrozen cells were thawed in distilled water at 36 °C for 45–60 s then enzymatically detached from mesh and added in re-warmed media with 15% FBS and washed twice at 1200 rpm for 5 minN/AN/AN/AN/A↔NS
Growth CurveCountess automated cell counterNRCanine BMN/A1921 × 104 cells/mlFrozen cells were thawed in distilled water at 36 °C for 45–60 s then enzymatically detached from mesh and added in re-warmed media with 15% FBS and washed twice at 1200 rpm for 5 minN/AN/AN/AN/A↔NS
Growth CurveCountess automated cell counterNRCanine BMN/A2161 × 104 cells/mlFrozen cells were thawed in distilled water at 36 °C for 45–60 s then enzymatically detached from mesh and added in re-warmed media with 15% FBS and washed twice at 1200 rpm for 5 minN/AN/AN/AN/A↔NS
Growth CurveCountess automated cell counterNRCanine BMN/A2401 × 104 cells/mlFrozen cells were thawed in distilled water at 36 °C for 45–60 s then enzymatically detached from mesh and added in re-warmed media with 15% FBS and washed twice at 1200 rpm for 5 minN/AN/AN/AN/A↔NS
Growth CurveCountess automated cell counterNRCanine BMN/A2641 × 104 cells/mlFrozen cells were thawed in distilled water at 36 °C for 45–60 s then enzymatically detached from mesh and added in re-warmed media with 15% FBS and washed twice at 1200 rpm for 5 minN/AN/AN/AN/A↔NS
Growth CurveCountess automated cell counterNRCanine BMN/A2881 × 104 cells/mlFrozen cells were thawed in distilled water at 36 °C for 45–60 s then enzymatically detached from mesh and added in re-warmed media with 15% FBS and washed twice at 1200 rpm for 5 minN/AN/AN/AN/A↔NS
Growth CurveCountess automated cell counterNRCanine BMN/A3121 × 104 cells/mlFrozen cells were thawed in distilled water at 36 °C for 45–60 s then enzymatically detached from mesh and added in re-warmed media with 15% FBS and washed twice at 1200 rpm for 5 minN/AN/AN/AN/A↔NS
CD 105 expressionAntibody assayNRCanine BMN/AOvernightNRPrimary antibodies (1:100 dilutions) were
used for localizing different markers (CD73, CD90, CD105, CD34) with
an overnight incubation period at 4 °C.		N/AN/AN/AN/A↔NS
CD 90 expressionAntibody assayNRCanine BMN/AOvernightNRPrimary antibodies (1:100 dilutions) were
used for localizing different markers (CD73, CD90, CD105, CD34) with
an overnight incubation period at 4 °C.		N/AN/AN/AN/A↔NS
CD 73 expressionAntibody assayNRCanine BMN/AOvernightNRPrimary antibodies (1:100 dilutions) were
used for localizing different markers (CD73, CD90, CD105, CD34) with
an overnight incubation period at 4 °C.		N/AN/AN/AN/A↔NS
Population Doubling TimeN/ANRCanine BMN/AN/A1 × 104 cells/mlN/AN/AN/AN/AN/A↔NS
Rogulska et al., 2019Metabolic Activity/Proliferation rateAlamar Blue3Human AdiposeN/A48NRMSCs culture in PS1D-based gelN/AN/AN/AN/AFresh betterP<0.05
Metabolic Activity/Proliferation rateAlamar Blue3Human AdiposeN/A96NRMSCs culture in PS1D-based gelN/AN/AN/AN/AFresh betterP<0.05
Metabolic Activity/Proliferation rateAlamar Blue3Human AdiposeN/A144NRMSCs culture in PS1D-based gelN/AN/AN/AN/A↔NS
ViabilityAlamar Blue3Human AdiposeN/A24NRN/AN/AN/AN/AN/AFresh betterP<0.05
Khan et al., 2019Antioxidant Concentration
(2 fresh groups:GFP-MSC and HO-1 MSC)		Antioxidant Assay6Canine adiposeNRNRNRLentivirus-mediated GFP and HO-1 gene insertion into Ad-MSCsN/AN/AN/AN/AFresh betterP<0.05
Yea et al., 2020ViabilityTrypan Blue6Human Umbilical Cord00, 2, 4, 24, 48 hr1×104 cells/wellNoneN/AN/AN/AN/A↔NS
ViabilityWater-soluble tetrazolium salt (WST) assay6Human Umbilical Cord00, 2, 4, 24, 48 hr1×10^4 cells/wellNoneN/AN/AN/AN/A↔NS
Population Doubling TimeCell counting6Human Umbilical Cord04, 8, 12, 16, 20 days3×10^3 cells/cm^2NoneN/AN/AN/AN/A↔NS
Horiuchi et al., 2021BiolumnescenceIVIS Lumina XRMS series III instrument (SPI, Tokyo,
Japan)		4Rat Synovial MSCs0Same dayVarying concentrationsNoneN/AN/AN/AN/A↔NS

  1. N/A = Not applicable (e.g. if the experiment set up did not include a particular variable). NR = Not reported (e.g. if a particular variable was part of the experiment set up but not explicitly reported on in results section or graph).

Table 7
Summary of all in vitro PBMC Proliferation assays from included studies.
StudyMSCs UsedSolutionAddition to solutionResponder CellsFresh vs. Frozen ComparisonDuration of CultureProliferation MeasurementRatio (MSC:Responder Cells)
1:11:31:61:101:121:50
Bárcia et al., 2017Cultured and Freshly-thawed MSCs were irradiated
with 50 Gy prior to use		RPMI5% HEPES, 5% Pen-Strep, 5% NaPyr and 5% human serumPBMC stimulated with anti-CD3, anti-CD28, and IL-2.Yes16 hrPercentage of T cells proliferation/
suppression		YesYesYes
Gramlich et al., 2016Cultured and Freshly-thawed MSCsRPMI10% (v/v) FBS, 1% (v/v) Penicillin/Streptomycin, and 1%
(v/v) L-glutamine		PBMC stimulated with 250,000 Human T-activator
CD3+/D28+Dynabeads		Yes144 hrCFSE Cell Proliferation KitYesYesYes
Tan et al., 2019Cultured and Freshly-thawed MSCsNRNRPBMC stimulated with Dynabeads Human T-Activator CD3/CD28Yes120 hrYes

Additional files

Supplementary file 1

Search strategy.

https://cdn.elifesciences.org/articles/75053/elife-75053-supp1-v1.docx
Supplementary file 2

PRISMA checklist.

https://cdn.elifesciences.org/articles/75053/elife-75053-supp2-v1.docx
Supplementary file 3

AGREE-II tool prompting questions.

https://cdn.elifesciences.org/articles/75053/elife-75053-supp3-v1.docx
Supplementary file 4

Data collection items.

https://cdn.elifesciences.org/articles/75053/elife-75053-supp4-v1.docx
MDAR checklist
https://cdn.elifesciences.org/articles/75053/elife-75053-mdarchecklist1-v1.pdf

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  1. Chintan Dave
  2. Shirley HJ Mei
  3. Andrea McRae
  4. Christine Hum
  5. Katrina J Sullivan
  6. Josee Champagne
  7. Tim Ramsay
  8. Lauralyn McIntyre
(2022)
Comparison of freshly cultured versus cryopreserved mesenchymal stem cells in animal models of inflammation: A pre-clinical systematic review
eLife 11:e75053.
https://doi.org/10.7554/eLife.75053