Ex vivo Induction of Apoptotic Mesenchymal Stem Cell by High Hydrostatic Pressure

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. Graphical Abstract

High Hydrostatic Pressure Therapy Annihilates Squamous Cell Carcinoma in a Murine Model

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

Exploration of the Pressurization Condition for Killing Human Skin Cells and Skin Tumor Cells by High Hydrostatic Pressure

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

Collagen/Gelatin Sponges (CGSs) Provide Both Protection and Release of bFGF: An In Vitro Study

It has been reported that collagen/gelatin sponges (CGSs) are able to sustain the release of basic fibroblast growth factor (bFGF) for approximately 10 days via the formation of ion complexes between bFGF and gelatin. CGSs impregnated with bFGF have been proven to promote dermis-like tissue formation in various in vivo studies and clinical trials. However, the bioactivities of bFGF released from CGSs have not been explored in vitro. In this study, we explored the ability of CGS impregnated with bFGF, stored at 37°C for up to 14 days, to promote fibroblast proliferation and the sustained release of bFGF. We analyzed the cellular viability and proliferation in 2D and in 3D cell cultures, by a CCK-8 assay. Furthermore, in order to characterize the morphological alteration of fibroblasts, we studied 3D cultures by microscopy with a scanning electron microscope (SEM) and a confocal microscope. Our analyses revealed that the fibroblasts were elongated and flanked each other. They infiltrated and migrated inside the CGSs and were oriented along the CGS structure. Thus, these data prove that CGSs protect and sustain the efficient release of growth factor for more than 7 days

Comparison of the efficacy of cryopreserved human platelet lysate and refrigerated lyophilized human platelet lysate for wound healing

Introduction: Human platelet lysate (hPL) part of the growth factor cocktail derived from human platelets, which has been applied as a cell growth supplement. The production process is easier in comparison to platelet-rich plasma; thus, hPL is now considered for use in wound healing therapy. However, methods for preserving hPL for more than several months that maintain its bioactivity must be considered, especially for chronic wound treatment. The present study compared the effects of preservation for 9 months using a refrigerator or deep freezer. Methods: We investigated three preservation conditions. In the C-hPL group, hPL was stored at −80 °C in a deep freezer for 9 months; in the CL-hPL group, hPL was cryopreserved for 9 months at −80 °C in a deep freezer then lyophilized; in the L-hPL group, lyophilized hPL was refrigerated at 4 °C for 9 months. The quantity and quality of growth factors in these three groups were measured by an ELISA and in fibroblast cell cultures. Then, gelatin hydrogel discs were impregnated with hPL and its effects with regard to the promotion of wound healing in mice were evaluated by histologic examinations. Results: The PDGF-BB concentration in C-hPL, CL-hPL and L-hPL was 18,363 ± 370 pg/ml, 11,325 ± 171 pg/ml, and 12,307 ± 348 pg/ml, respectively; the VEGF concentration was 655 ± 23 pg/ml, 454 ± 27 pg/ml, and 499 ± 23 pg/ml, respectively; and the TGF-β1 concentration was 97,363 ± 5418 pg/ml, 73,198 ± 2442 pg/ml, and 78,034 ± 3885 pg/ml, respectively. In cell culture medium, fibroblast cell cultures were better supported in the hPL groups than in the fetal bovine serum group. In the histologic examination of the wound healing process, no differences were observed among the three preserved hPL groups with regard to epithelialization, or granulation tissue or capillary formation. The wounds in all groups had almost healed by day 14. Conclusions: The stability of growth factors contained in lyophilized hPL is maintained at 4 °C for up to 9 months. This was a versatile preservation method that can be applied in clinical practice. Keywords: Platelet lysate, Gelatin hydrogel, Cryopreservation, Lyophilization, Long-term preservation, Wound healin

Hydrostatic pressure can induce apoptosis of the skin

Abstract We previously showed that high hydrostatic pressure (HHP) treatment at 200 MPa for 10 min induced complete cell death in skin and skin tumors via necrosis. We used this technique to treat a giant congenital melanocytic nevus and reused the inactivated nevus tissue as a dermis autograft. However, skin inactivated by HHP promoted inflammation in a preclinical study using a porcine model. Therefore, in the present study, we explored the pressurization conditions that induce apoptosis of the skin, as apoptotic cells are not believed to promote inflammation, so the engraftment of inactivated skin should be improved. Using a human dermal fibroblast cell line in suspension culture, we found that HHP at 50 MPa for ≥ 36 h completely induced fibroblast cell death via apoptosis based on the morphological changes in transmission electron microscopy, reactive oxygen species elevation, caspase activation and phosphatidylserine membrane translocation. Furthermore, immunohistochemistry with terminal deoxynucleotidyl transferase dUTP nick-end labeling and cleaved caspase-3 showed most cells in the skin inactivated by pressurization to be apoptotic. Consequently, in vivo grafting of apoptosis-induced inactivated skin resulted in successful engraftment and greater dermal cellular density and macrophage infiltration than our existing method. Our finding supports an alternative approach to hydrostatic pressure application