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Among promising solutions for tissue repair and wound healing, mesenchymal stem (or stromal) cells (MSCs) have been a focus of attention and have become the most clinically studied experimental cell therapy. Recent studies reported the importance of apoptosis in MSC-mediated immunomodulation, in which apoptotic MSCs (apoMSCs) were shown to be superior to living MSCs. Nowadays, high hydrostatic pressure (HHP), a physical technique that uses only fluid pressure, has been developed and applied in various bioscience fields, including biotechnology, biomaterials, and regenerative medicine, as its safe and simply operation. In the current study, we investigated the impact of HHP treatment on human bone marrow-MSC survival and proliferation. Based on the detection of executioner caspase activation, phosphatidylserine exposure, DNA fragmentation (TUNEL) and irrefutable ultrastructural morphological changes on transmission electron microscopy (TEM), our data revealed that HHP treatment induced complete apoptosis in MSCs. Notably, this technique might provide manipulated products for use in cell-based therapies as manufacturing capability expands. We hope that our findings will contribute to the improvement of MSCs or EVs in translational research development.

High hydrostatic pressure (HHP) is a physical method for inactivating cells or tissues without using chemicals such as detergents. We previously reported that HHP at 200 MPa for 10 min was able to inactivate all cells in skin and giant congenital melanocytic nevus (GCMN) without damaging the extracellular matrix. We also reported that HHP at 150 MPa for 10 min was not sufficient to inactivate them completely, while HHP at 200 MPa for 10 min was able to inactivate them completely. We intend to apply HHP to treat malignant skin tumor as the next step; however, the conditions necessary to kill each kind of cell have not been explored. In this work, we have performed a detailed experimental study on the critical pressure and pressurization time using five kinds of human skin cells and skin tumor cells, including keratinocytes (HEKas), dermal fibroblasts (HDFas), adipose tissue-derived stem cells (ASCs), epidermal melanocytes (HEMa-LPs), and malignant melanoma cells (MMs), using pressures between 150 and 200 MPa. We pressurized cells at 150, 160, 170, 180, or 190 MPa for 1 s, 2 min, and 10 min and evaluated the cellular activity using live/dead staining and proliferation assays. The proliferation assay revealed that HEKas were inactivated at a pressure higher than 150 MPa and a time period longer than 2 min, HDFas and MMs were inactivated at a pressure higher than 160 MPa and for 10 min, and ASCs and HEMa-LPs were inactivated at a pressure higher than 150 MPa and for 10 min. However, some HEMa-LPs were observed alive after HHP at 170 MPa for 10 min, so we concluded that HHP at a pressure higher than 180 MPa for 10 min was able to inactivate five kinds of cells completely.

Background: Apoptosis was reported to take crucial role in mesenchymal stem cell (MSC)-mediated immunomodulation, in which apoptotic MSCs were shown to be superior compared to living MSCs. Furthermore, extracellular vesicles (Evs) derived from MSCs were revealed more specific advantages for patient safety such as lower propensity to trigger innate and adaptive immune responses. As a safety and simple operation, high hydrostatic pressure (HHP), a physical technique that uses only fluid pressure to inactivate cells or tissues, has been developed and applied in a lot of field of biosciences, including biotechnology, biomaterials, or tissue engineering. Methods: MSCs isolated from human bone marrow were suspended cultured in appropriate medium and subjected to pressurization at 50 MPa for 36 h. Then cells were collected and investigated apoptotic pathway by transmission electron microscopy (TEM), phosphatidylserine membrane translocations, cleaved caspase-3/7 and terminal deoxy-nucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. Besides, viability assays and live cell imaging were also used for assessement of cell survival after pressurization. Results: We found that HHP at 50 MPa for ≥36 h completely induced MSC death by Live/Dead assay, live cell imaging and WST-8 assay up to 7 days after pressurization. The large amount of apoptotic MSCs death was found based on morphological changes in TEM, phosphatidylserine exposure, caspase activation and detection of DNA fragmentations via TUNEL staining. Conclusions: In the current study, our data revealed that HHP treatment was convenient processing which safety and effectively induced MSCs undergo apoptosis. Especially, by capable of manufacture expanding, this technique might provide numbers of manipulated products using for industrial cell-based therapies.

Cutaneous squamous cell carcinoma (cSCC) is one of the most common skin cancers. In the treatment of cSCC, it is necessary to remove it completely, and reconstructive surgery, such as a skin graft or a local or free flap, will be required, depending on the size, with donor-site morbidity posing a burden to the patient. The high hydrostatic pressure (HHP) technique has been developed as a physical method of decellularizing various tissues. We previously reported that HHP at 200 MPa for 10 min could inactivate all cells in the giant congenital melanocytic nevus, and we have already started a clinical trial using this technique. In the present study, we explored the critical pressurization condition for annihilating cSCC cells in vitro and confirmed that this condition could also annihilate cSCC in vivo. We prepared 5 pressurization conditions in this study (150, 160, 170, 180, and 190 MPa for 10 min) and confirmed that cSCC cells were killed by pressurization at ≥160 MPa for 10 min. In the in vivo study, the cSCC cells inactivated by HHP at 200 MPa for 10 min were unable to proliferate after injection into the intradermal space of mice, and transplanted cSCC tissues that had been inactivated by HHP showed a decreased weight at 5 weeks after implantation. These results suggested that HHP at 200 MPa for 10 min was able to annihilate SCC, so HHP technology may be a novel treatment of skin cancer.

Pelnac GplusⓇ, IntegraⓇ, and TerudermisⓇ are approved artificial dermis products in Japan. Previously, we proved that Pelnac GplusⓇ was able to sustain basic fibroblast growth factor (bFGF) and accelerated wound healing by releasing impregnated bFGF. In this study, we impregnated Pelnac GplusⓇ, IntegraⓇ, and TerudermisⓇ with bFGF and compared the binding activity and wound-healing process. We applied bFGF to each material and compared the bFGF concentrations in the surrounding area after 24-h incubation. For the in vivo study, dermal substitutes were impregnated with bFGF and implanted into full-thickness wounds of BKS.Cg-+Leprdb/+Leprdb/Jcl mice. Wounds were evaluated at days 7, 14, and 21 after implantation. The in vitro study showed that bFGF is strongly bound to IntegraⓇ, followed by Pelnac GplusⓇ and TerudermisⓇ. The in vivo study showed that fibroblasts and capillaries had infiltrated into the Pelnac GplusⓇ but not the IntegraⓇ or TerudermisⓇ. Furthermore, long epithelium and wide granulation tissue were formed in the Pelnac GplusⓇ with bFGF group. The TerudermisⓇ with bFGF group had more capillaries than other groups, but only at the base of the wound. The combination of Pelnac GplusⓇ with bFGF may be a novel approach for treating full-thickness skin defects or chronic skin ulcers.

Background The efficacy of a gelatin hydrogel (GH) sheet impregnated with platelet-rich plasma in full-thickness wound healing has been reported. Human platelet lysate is another potential natural product for use in wound healing. The present study examined the effects of a GH sheet impregnated with concentrated freeze-dried platelet lysate on wound healing after storage for 9 mo. Materials and methods Platelet concentrates were subjected to three freeze-thaw cycles and freeze-dried then preserved at 4°C. Reconstitution with saline was then performed to produce 1-fold (hPL1), 2-fold (hPL2), and 3-fold (hPL3) concentrations of preserved platelet lysate. Full-thickness wounds were made on the back of male C57Bl6J/Jcl mice. Wounds were treated with saline, hPL1, or a GH sheet impregnated with saline, hPL1, hPL2, or hPL3. Histologic examinations using hematoxylin–eosin, Azan, and anti-CD31 staining were performed on days 4, 7, and 14 to assess neoepithelialization and granulation tissue and capillary formation. Results This study showed that the GH sheet itself or the simple administration of hPL1 did not accelerate the healing process. However, the GH sheet impregnated with hPL1 accelerated the granulation tissue formation to some extent, and the GH sheet impregnated with hPL2 or hPL3 clearly accelerated the capillary formation and the granulation tissue formation. In addition, the GH sheet impregnated with hPL3 had the longest epithelium formation. Conclusions A GH sheet impregnated with long-term preserved 2-fold or 3-fold concentrated platelet lysate enhances the wound healing process.

In this work, the effects of sintering temperature and Na subtitution on zero critical temperature (Tc,0) in Bi-Pb-Sr-Ca-Cu-O superconductors were investigated. The Bi1.6Pb0.4Sr2Ca2-xNaxCu3O10+d bulk samples with x = 0.00 and 0.05 (BPSCCO) were prepared by using the solid state reaction technique at elevated sintering temperatures of 840 and 850oC. To examine the improvements of structural and superconducting properties of the BPSCCO samples, the X-ray diffraction (XRD), scanning electronic microscope (SEM) and DC resistance versus temperature (R-T) measurements were carried out. The experimental findings exhibit a co-existence of the high Tc (Bi-2223) and low Tc (Bi-2212) phases in all samples. The estimated volume fraction of the Bi-2223 phase was increased from 71.4 to 75.7%, and the average size of the Bi-2223 grains was enlarged from 4.5 to 9.8 mm as changing the sample preparation conditions. The improved connectivities between the Bi-2223 grains were also obtained by quantitively analyzing the relation between Tc,0 and residual resistance ratio (RRR). These improvements were likely to be reasons for the enhancements of Tc,0 in the BPSCCO samples.