Human primary fibroblasts were established and stored in liquid nitrogen [16] . Human primary fibroblasts were recovered from liquid nitrogen and cultured in alpha MEM (GIBCO, Carlsbad, CA, USA) containing 5% human platelet lysate (HPL) (Mill Creek Life Sciences, Rochester, MN, USA). Human MSCs were derived from bone morrow and obtained from RoosterBio (Frederick, MD, USA). MSCs were recovered from liquid nitrogen and cultured in the complete Rooster media (Frederick, MD, USA). NSC-34 is a hybrid cell line, produced by fusion of motor neuron enriched, embryonic mouse spinal cord cells with mouse neuroblastoma. NSC-34 was purchased from Cedarlane Corporation (Ontario, Canada) and cultured in Dulbecco’s modified eagle medium (DMEM) (GIBCO, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) [16] . When cells grew to 80% full in flask, they were digested with TrypLE expression solution (GIBCO, Carlsbad, CA, USA) and sub-cultured at 37˚C and 5% CO₂.

Mitochondrial membrane potential (MMP) generated by proton pumps is an essential component in the process of energy storage during OXPHOS. Membrane potential dependent dyes such as JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolocarbocyanine iodide) and MitoTracker dyes have been used to stain mitochondria and monitor mitochondrial potential. For JC-1 staining, mitochondria were stained with mitochondria staining kit (Sigma CS0390, St. Louis, MO, USA). The protocol was referred to the document of the product’s manufacturer. Fibroblasts and MSCs were incubated with JC-1 solution for 20 minutes at 37˚C in humidified atmosphere containing 5% CO₂. Fluorescence was observed by Olympus IX83 fluorescent microscope. In cells which maintained electrochemical potential gradient, the dye concentrates in mitochondria, where it formed bright red fluorescent aggregates (J-aggregates). If cells failed to maintain MMP, the JC-1 was dispersed through the entire cells resulting in a shift from red to green fluorescence (JC-1 monomers). Cells were treated with mitochondrial inhibitors, valinomycin or FCCP. The treated cells were controls of mitochondrial dissipation.

Mitochondrial isolation followed a previously described protocol [16] . All reagents were sterile. 50 × 10⁶ Fibroblasts or MSCs were centrifuged for 5 minutes at 400 g and at 4˚C to remove the media. Cell pellet was re-suspended in ice-cold 300 mM sucrose mitochondrial isolation buffer (MIB) (Sigma Aldrich, St. Louis, MO, USA) and homogenized by bead beating (Bead Ruptor 12, Omni International homogenizer company, Kennesaw, GA, USA). The cell lysate was centrifuged for 10 minutes at 700 g and at 4˚C. Then the supernatant was transferred to new centrifugation tubes and centrifuged for 10 minutes at 9000 g at 4˚C. The supernatant was removed. The wet weight of mitochondria was measured [16] . The mitochondrial pellet was re-suspended with 240 mM sucrose mitochondrial respiration buffer (MRB) (Sigma Aldrich, St. Louis, MO, USA). Mitochondrial suspension was cooled in wet ice. MMP and ATP content of the mitochondria were stable for 24 hours. MMP and mitochondrial ATP content were measured within 2 hours after isolation in the current study.

The stock solution of JC-1 was added to mitochondrial suspension to a final concentration 1 µg/ml. The mixture was incubated for 10 minutes at room temperature. Isolated mitochondria were treated with mitochondrial inhibitor valinomycin or FCCP. The treated mitochondria were controls of mitochondrial dissipation. Red fluorescent J-aggregates in intact mitochondria could be observed under fluorescent microscope. The relative fluorescence units (RFU) could be read in multiple plate fluorimeter using end-point method with the setting of Ex/Em: 490 nm/590 nm.

ATP content was measured with ATPlite kit (Perkin Elmer Inc., Waltham, MA, USA). The detailed procedure was referred to the product manual. The brief method was as follows: added 50 µl of mammalian cell lysis solution to 100 µl of fibroblasts or MSCs, mitochondria, or MRB per well in a 96-well plate with white well and clear bottom; shook the plate for 5 minutes; added 50 µl substrate solution to all wells and shook the plate for 5 minutes; measured the luminescence of the plate; calculated ATP content of samples using the ATP standard curve.

The respiration assay buffer (mitochondrial assay solution; MAS) contains 70 mM sucrose, 220 mM mannitol, 10 mM KH₂PO₄, 5 mM MgCl₂, 2 mM HEPES, and 1 mM EGTA. For carrying out respiration using isolated mitochondria, pyruvate (10 mM) and malate (5 mM) were added to the MAS, and the resulting solution used to make 10× stocks of the respiratory inhibitors and uncouplers. The respiratory stocks were loaded into the drug ports of a hydrated sensor cartridge in the following order: (A) oligomycin (2.5 µg/mL final), (B) FCCP (4 µM final), and (C) antimycin A (4 µM final) + rotenone (2 µM final). The protein concentrations of isolated mitochondria preparations were measured. Equal amounts of mitochondria (1 µg protein/well) were plated on the Seahorse cell culture microplate in 20 µL of MAS + substrate + 0.2% w/v fatty-acid free BSA and centrifuged at 2000×g for 20 min at 4˚C. The assay medium (MAS + substrates + 0.2% BSA + 4.5 mM ADP) was then added to the wells to bring the final volume to 180 µL prior to the plate being incubated at 37˚C for 30 min and transferred to the analyzer for analysis. The respiration assay protocol consisted of a minimum of three cycles of OCR measurements for each measurement period. Each cycle consisted of a 2 min “mix” period and 2 min “wait” period, followed by a 3 min “measure” period. Three cycles were used to obtain a basal OCR, 6 cycles were used to assess the effect of the F1-Fo ATP synthase inhibitor oligomycin, three cycles were used to evaluate the effect of the uncoupler FCCP, and 3 cycles were used to measure mitochondria-associated respiration following injection of antimycin A/rotenone.

Cryogenically frozen MSCs were quickly thawed by gently swirling the vials in the 37˚C water bath. Mitochondria of the recovered MSCs or fresh MSCs were labelled using 150nM of MitoTracker Red dye (Thermofisher Scientific, Waltham, MA, USA) at 37˚C and 5% CO₂ for 30 minutes. The cells were washed with Hanks’ balanced salt solution (HBSS) 3 times to remove the dye. Then, mitochondria were isolated. The MitoTracker Red-labelled mitochondria were added to NSC-34 cells that grew on glass surface in glass-bottom culture dishes and incubated overnight at 37˚C and 5% CO₂. Then the NSC-34 cells were washed 3 times with pre-warmed HBSS to remove the labelled mitochondria in the media. The cell dishes were observed under fluorescent microscope.

Student’s t-test was used to test statistical significance by GraphPad Prism. p-value less than 0.05 was judged to be of statistical significance.

Cryovials containing frozen fibroblasts or MSCs were removed from liquid nitrogen storage and immediately placed into a 37˚C water bath. Cells were quickly thawed by gently swirling the vials in the 37˚C water bath until there was just a small bit of ice left in the vials. The vials were transferred to a laminar flow hood. MMP was shown by JC-1 staining. Red fluorescence was observed in mitochondria of the fibroblasts and MSCs. Red fluorescent brightness and distribution in the cryopreserved cells were not different from the growing fibroblasts and MSCs that attached on surface of slides ( Figure 1 , Figure 2 ). Valinomycin or FCCP-treated cells lost most red fluorescence. The data showed that cryogenically frozen fibroblasts and MSCs in liquid nitrogen maintained similar MMP to the fresh fibroblasts and MSCs.

Vials containing 50 × 10⁶ frozen MSCs in liquid nitrogen or dry ice were immediately placed into a 37˚C water bath by gently swirling the vials until there was just a small bit of ice left in the vials. Mitochondria were isolated from the MSCs and proceeded to measurement of MMP and ATP content. The mitochondria from the cryopreserved MSCs actively took up dye JC-1 and formed bright red fluorescence (J-aggregates) that was similar to the mitochondria isolated fresh MSCs ( Figure 3 ). In mitochondrial suspension with concentration 100 mg/ml, RFU of the mitochondria of cryopreserved MSCs (435 ± 45) was slightly lower than the mitochondria from fresh MSCs (488 ± 82), but the difference was not significant (p > 0.05).

In agreement with MMP, ATP content of the mitochondria from frozen MSCs (51.8 ± 7.7 pmol/mg mitochondria) was slightly less than the mitochondria from fresh MSCs (64.8 ± 8.9 pmol/ml), but the difference was not significant (p > 0.05). These results of MMP and ATP content showed that cryopreservation maintained mitochondrial function of MSCs and cryopreserved MSCs were an alternative mitochondrial source to fresh MSCs.

We reported previously that isolated mitochondria of human fibroblasts transferred to NSC34 cells after co-culture [16] . After 16 hours of co-culture, MitoTracker Red-labelled mitochondria of MSCs were seen in NSC-34 cells under fluorescent microscope. Mitochondria from both fresh and cryopreserved MSCs could enter to NSC-34 cells ( Figure 4 ). The results showed that mitochondria from cryopreserved MSCs had similar ability to transfer into NSC-34 cells to mitochondria of fresh MSCs.

Morphological characteristics of fibroblasts and MSCs are alike ( Figure 5 ). Both fibroblasts and MSCs have abundant mitochondria indicated by membrane potential dependent dye JC-1 ( Figure 1 , Figure 2 ). However, MSCs demonstrated a higher level of ATP than fibroblasts (all were significant at p < 0.01, at 1250, 2500,

5000 and 10,000 cells per wells) ( Figure 6 ). We measured MMP and ATP content of mitochondria isolated from fresh fibroblasts and MSCs. We found MMP was not significantly different between the isolated mitochondria of fibroblasts (RFU: 548 ± 108) and MSCs (RFU: 488 ± 82) (p > 0.05) ( Figure 7 ). In alignment with the ATP content results in whole fibroblasts and MSCs, the isolated mitochondria from MSCs (64.8 ± 8.9 pmol/mg mitochondria) had significantly higher ATP content than the mitochondria of fibroblasts (24.5 ± 1.8 pmol/ml) (p < 0.01) ( Figure 8 ). Finally, we measured the OCR in the isolated mitochondria from fresh and frozen cells to determine the impact on respiration. Both fresh and frozen fibroblasts (9a) and MSCs (9b) showed similar patterns of OCR and the frozen cells did not show any compromised effect of the cell freezing ( Figure 9 ). In addition, as seen in Figure 6 and Figure 8 , because of the increased ATP levels in the MSC cells the OCR was higher in the MSC cells as well.