There are many ways to Determine the Exact Number and Viability of Stem Cells in Umbilical Cord Blood or Stromal Vascular Fraction, the problem is that most all of them are expensive and time consuming.
Here is a paper which shows a rapid and accurate method, but it is still is involved and not cheap per patient use: https://www.fishersci.com/shop/products/promega-cytotox-glo-cytotoxicity-assay-3/p-3573963
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Here is a paper which shows a rapid and accurate method, but it is still is involved and not cheap per patient use: https://www.fishersci.com/shop/products/promega-cytotox-glo-cytotoxicity-assay-3/p-3573963
Looking into more ways to rapidly and accurately determine stem cell amounts and viability in SVF or any adipose sample.
To prevent cryoinjuries, it is necessary to establish the optimal cooling rate and concentration of cryoprotectant, which represent 2 main factors for the survival of frozen cells. The best cell recovery is after controlled freezing with 5–10% dimethyl-sulphoxide (DMSO), but a unique, accepted, and standardized cryopreservation protocol has not yet been established.8
The viability of thawed hematopoietic progenitor cells is traditionally evaluated with the trypan blue exclusion test. Viable cells exclude trypan blue stain uptake, due to their intact cell membranes; nonviable cells, however, are membrane-porous cells which stain blue. This method requires a technician, a hemocytometer, and a light microscope to enumerate both stained and unstained cells. The percentage viability is then calculated.9
Apart from the manual method, there is an automated system for viability evaluation, using a PC and a Charge-Couple Device (CCD) camera connected to a machine that uses trypan blue for the automated enumeration of dead and viable cells.9
Recently, a novel luminescent system was developed to evaluate the cytotoxicity of several cytotoxic agents. There are several different kits that can be used with the Glo-Max system including the CytoTox-Glo Cytotoxicity Assay. This is a luminescent cytotoxicity assay that measures the relative number of dead cells in cell populations. The CytoTox-Glo Assay measures the extracellular activity of a distinct intracellular protease (dead-cell protease) when the protease is released from membrane-compromised cells. A luminogenic cell-impermeant peptide substrate (AAF-aminoluciferin) is used to measure dead-cell protease activity. The released aminoluciferin product is measured as “glow type” luminescence generated by Ultra-Glo Recombinant Luciferase provided in the assay reagent. The AAF-aminoluciferin substrate cannot cross the intact membrane of viable cells and does not generate any appreciable signal from the live-cell population. The amount of luminescence correlates directly with the percentage of cells undergoing cytotoxic stress. With the addition of a lysis reagent, the Cyto-Tox-Glo Assay can also deliver the luminescent signal associated with the total number of cells in each assay well. Viability can be calculated by subtracting the luminescent dead-cell signal from the total luminescent value, thus allowing you to normalize assay data to cell number and mitigate assay interferences that may lead to erroneous conclusions.10–12
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Published online 2016 Jan 6. doi: 10.1155/2016/9240652
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Figure 1
Procedure for SCT. Cells can be labeled with contrast agent either directly or indirectly. The labeled cells are purified from unlabeled cells to obtain a cell product with high signal and thus contrast versus adjacent tissue. Before the delivery, the stability of the labeled cells can be tested to assess any potential toxicity of the imaging agent. After delivery, the viability of the delivered cells is monitored to understand engraftment and survival. The labeled stem cells can be clearly recognized due to increased signal produced by the label. Finally, histology and associated microscopy techniques can confirm that the imaging signal does indeed correspond to the cells of interest.
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Kotaro Yoshimura
Kotaro Yoshimura received his medical degree from the University of Tokyo School of Medicine, Tokyo, Japan, in 1985. After completion of his residency and fellowship in plastic surgery, he was board certified in plastic surgery by the Japanese Society of Plastic and Reconstructive Surgery in 1990. He began studying physiology of peripheral nerve and skeletal muscle in 1991and studied at the University of Michigan from 1994 to 1994. He was assigned as a faculty member at the University of Tokyo School of Medicine in 1995. He was assigned as an Associate Professor at the Department of Plastic Surgery at the University of Tokyo in 1998. Thereafter, his research interests have been tissue engineering and regenerative medicine for plastic, reconstructive, and aesthetic purposes. In particular, Dr. Yoshimura has studied on wound healing, angiogenesis, and adipogenesis with adipose-derived stem cells as well as hair regeneration using epithelial stem cells and dermal papilla cells.
Biography Updated on 20 May 2010
Scholarly Contributions [Data Provided by ]
- An injectable non-cross-linked hyaluronic-acid gel containing therapeutic spheroids of human adipose-derived stem cells, Scientific Reports, vol. 7, no. 1, pp. , 2017.
- Studying the blood pressures of antegrade and retrograde internal mammary vessels: Do they really work as recipient vessels?, Journal of Plastic, Reconstructive and Aesthetic Surgery, vol. 70, no. 10, pp. 1391–1396, 2017.
- Free-flap surgical correction of facial deformity after anteromedial maxillectomy, Journal of Cranio-Maxillofacial Surgery, vol. 45, no. 9, pp. 1573–1577, 2017.
- Pathological changes of adipose tissue in secondary lymphoedema, British Journal of Dermatology, vol. 177, no. 1, pp. 158–167, 2017.
- Reply: Mechanical Micronization of Lipoaspirates: Squeeze and Emulsification Techniques, Plastic and Reconstructive Surgery, vol. 139, no. 6, pp. 1370e–1371e, 2017.
- Orbitomaxillary Reconstruction Using a Combined Latissimus Dorsi Musculocutaneous and Scapular Angle Osseous Flap, Journal of Oral and Maxillofacial Surgery, vol. 75, no. 2, pp. 439.e1–439.e6, 2017.
- Blood Congestion Can Be Rescued by Hemodilution in a Random-Pattern Skin Flap, Plastic and Reconstructive Surgery, vol. 139, no. 2, pp. 365–374, 2017.
- Mechanical Micronization of Lipoaspirates: Squeeze and Emulsification Techniques, Plastic and Reconstructive Surgery, vol. 139, no. 1, pp. 79–90, 2017.
- Laboratory research in plastic surgery: From study design to publication, Japanese Journal of Plastic Surgery, vol. 59, no. 12, pp. 1264–1270, 2016.
- Abdominal morbidity after single- versus double-pedicled deep inferior epigastric perforator flap use, Journal of Plastic, Reconstructive and Aesthetic Surgery, vol. 69, no. 9, pp. 1178–1183, 2016.
- Lipoinjection – Past progress and future perspectives, Japanese Journal of Plastic Surgery, vol. 59, no. 5, pp. 514–523, 2016.
- Breast reconstruction by multistep cell-assisted lipotransfer (step-CAL) after mastectomy for breast cancer, Japanese Journal of Plastic Surgery, vol. 59, no. 5, pp. 496–504, 2016.
- Therapeutic potential of human adipose-derived stem/stromal cell microspheroids prepared by three-dimensional culture in non-cross-linked hyaluronic acid gel, Stem Cells Translational Medicine, vol. 4, no. 12, pp. 1511–1522, 2015.
- Clinicopathologic Assessment of Myositis Ossificans Circumscripta of the Masseter Muscles, Journal of Craniofacial Surgery, vol. 26, no. 6, pp. 2025–2026, 2015.
- Future Perspectives of Fat Grafting, Clinics in plastic surgery, vol. 42, no. 3, pp. , 2015.
- Fat Grafting: Current Concept, Clinical Application, and Regenerative Potential, Part 2. Preface, Clinics in plastic surgery, vol. 42, no. 3, pp. xiii–xiv, 2015.
- Vibration inhibits deterioration in rat deep-tissue injury through HIF1-MMP axis, Wound Repair and Regeneration, vol. 23, no. 3, pp. 386–393, 2015.
- How does fat survive and remodel after grafting?, Clinics in Plastic Surgery, vol. 42, no. 2, pp. 181–190, 2015.
- Condensation of tissue and stem cells for fat grafting, Clinics in Plastic Surgery, vol. 42, no. 2, pp. 191–197, 2015.
- Establishment of a novel rat model for deep tissue injury deterioration, International Wound Journal, vol. 12, no. 2, pp. 202–209, 2015.
- Treatment for facial pigmented skin lesions, Japanese Journal of Plastic Surgery, vol. 58, no. 1, pp. 23–31, 2015.
- Complications of Fat Grafting: How They Occur and How to Find, Avoid, and Treat Them, Clinics in Plastic Surgery, vol. 42, no. 3, pp. 383–388, 2015.
- Fat grafting: Current concept, clinical application, and regenerative potential, part 1, Clinics in Plastic Surgery, vol. 42, no. 2, pp. ix–x, 2015.
- A novel facial rejuvenation treatment using pneumatic injection of non-cross-linked hyaluronic acid and hypertonic glucose solution, Dermatologic Surgery, vol. 41, no. 6, pp. 755–758, 2015.
- Future Perspectives of Fat Grafting, Clinics in Plastic Surgery, vol. 42, no. 3, pp. 389–394, 2015.