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

Toshihito Mitsui,Naoki Morimoto,Natsuko Kakudo,Tien Minh Le,Sharon Claudia Notodihardjo,Mariko Moriyama,Atsushi Mahara,Maria Chiara Munisso,Tetsuji Yamaoka,Hiroyuki Moriyama,Kenji Kusumoto

doi:10.1155/2020/9478789

Abstract

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.

Highlights

High hydrostatic pressure (HHP) is a safe method of physically inactivating cells or tissues promptly without using chemicals, such as detergents, that is commonly used to prepare decellularized tissues
The area stained by the red fluorescence derived from ethidium homodimer II nucleic acid stain increased with HHP at any pressure for 2 min, with most of the cells stained red at 10 min (Figure 1(a))
[10], and we showed that porcine skin [2, 4, 6] as well as human skin and human nevus tissue were completely inactivated by HHP at 200 MPa for 10 min [1, 3, 5]

Summary

Introduction

High hydrostatic pressure (HHP) is a safe method of physically inactivating cells or tissues promptly without using chemicals, such as detergents, that is commonly used to prepare decellularized tissues. HHP at 200 MPa did not damage the extracellular matrix, HHP at 1000 MPa damaged and altered the epidermal basement membrane to some degree and prevented the survival of human cultured epidermis (hCE) on the pressurized skin or nevus [1]. We reported that autologous dermis pressurized at 200 MPa without removing cellular debris showed less contracture after grafting than decellularized allogeneic dermis using a porcine model [2]. Our exploratory clinical study to investigate the safety and efficacy of a novel treatment combining autologous nevus tissue inactivated by HHP at 200 MPa and a patient’s cultured epidermal autograft (CEA) to reconstruct skin defects after removal is ongoing. Our previous studies indicated that HHP at 150 MPa for 10 min was not sufficient to inactivate cells completely, while HHP at MPa for 10 min was able to inactivate cells completely

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