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.


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. We previously reported that HHP at 200 MPa for 10 min was able to inactivate all cells in porcine skin, human skin, and human nevus tissue (giant congenital melanocytic nevus (GCMN), representative of human congenital skin tumor) [1][2][3][4][5][6]. Furthermore, HHP at 200 MPa did not damage the extracellular matrix, although 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]. In addition, 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].
At present, 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 200 MPa for 10 min was able to inactivate cells completely. In this work, we intend to apply HHP to treat malignant skin tumor; however, the conditions necessary to kill each cell type have not yet been explored. In fact, regarding the death pathway after HHP, it was reported that cells after HHP at 200 MPa died through apoptosis, while those pressurized at >300 MPa died immediately following a necrotic pathway [7,8]. However, the death pathway taken under our HHP conditions has not yet been explored.
In the present study, we explored 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). 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 these five kinds of cells, we evaluated the pressurization condition that killed each cell type completely using the live/dead assay, morphological observation of the cultured cells, and the water-soluble terzolium salts (WST) assay. We then evaluated the death pathway using an apoptosis assay and transmission electron microscopy (TEM) as described below.

Live/Dead Staining of Cells without Pressurization or after
Pressurization. We prepared the live/dead staining working solution (Live/Dead Reduced Biohazard Viability/Cytotoxicity Kit #1; Life Technologies Co., Ltd.) by mixing Component A (SYTO10) and Component B (Ethidium homodimer II (EthD-II)) according to the manufacturer's instructions.
Cryopreserved HEKas, HDFas, ASCs, HEMa-LPs, and MMs were rapidly thawed in a water bath, and 1 × 10 6 cells each were seeded and cultured on a 10 or 15 cm culture dish with the respective culture medium. After reaching subconfluence, the cells were dissociated using TrypeLE ™ Express, and cell suspensions of 1 × 10 6 cells/ml in the respective culture medium were prepared. A total of 200 μl of each cell suspension was then packed in a plastic bag, and 16 bags were prepared for each kind of cell. One bag of each cell type was preserved at room temperature without pressurization in our safety cabinet (control sample), and the other 15 were pressurized under different conditions. We prepared 15 kinds of pressurization conditions in this study: pressurization at 150, 160, 170, 180, and 190 MPa and pressurization for 1 s, 2 min, and 10 min, respectively, at each pressure. In brief, each bag was placed in a sample chamber of an isostatic pressurization machine (ECHIGO SEIKA, Co., Ltd., Nagaoka, Japan), and the chamber was filled with tap water. e pressure was increased up to the target pressure of 150, 160, 170, 180, or 190 MPa. e target pressure was then maintained for 1 s, 2 min, or 10 min, after which the pressure was reduced. e suspensions of all groups, including the nonpressurization group (control sample), were then moved to 1.5 ml microtubes (Scientific Specialties, Inc., Lodi, CA, USA) and centrifuged at 200g for 5 min. e medium was discarded, and 200 µL of the working solution of live/dead staining was added to resuspend the cells.
is suspension was incubated for 15 min in the dark and then centrifuged at 200g for another 5 min. e medium was discarded, and the cells were then suspended again using 200 µL of the 0.6% paraformaldehyde solution and incubated for at least 15 min in the dark. After this, fluorescence micrographs were taken using a fluorescence microscope (BZ-9000; Keyence Corp., Osaka, Japan).

Morphological Observation and a Proliferation Assay of the Cells after Pressurization.
Cryopreserved HEKas, HDFas, ASCs, HEMa-LPs, and MMs were rapidly thawed, and 1 × 10 6 cells each were seeded on a 10 or 15 cm culture dish and cultured until reaching subconfluence, as mentioned above. Cells were dissociated using TrypeLE ™ Express, and cell suspensions of 1 × 10 5 cells/ml in the respective culture medium were prepared. A total of 5 ml of each cell suspension was then packed in a plastic bag, and 18 bags were prepared for each kind of cell. ree bags were preserved at room temperature without pressurization in our safety cabinet (control sample), and the other 15 were pressurized under different conditions.
We prepared 15 pressurization conditions, and each bag was pressurized as mentioned above. A 100 µL aliquot of each cell suspension, including the nonpressurization group, was then seeded into the well of a 24-well cell culture plate (CORNING, Inc., Corning, NY, USA) with 1 ml of the respective culture medium. Cells were cultured at 37°C, 95% humidity, and 5% carbon dioxide for 7 days without changing the medium. e cell morphology and attachment were observed at 3 h, 1 day, 3 days, and 7 days after seeding using the inverted microscope (Carl Zeiss Co., Ltd., Oberkochen, Germany).
In addition, the proliferation was evaluated quantitatively using a WST-8 (4-(3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio)-1, 3-benzene disulfonate sodium salt) assay (Cell Counting Kit-8; Dojindo, Kumamoto, Japan). In brief, a 100 μL aliquot of each cell suspension, either after pressurization or without pressurization, was added to each well (n � 36 per group of HEKas, HDFas, ASCs, HEMa-LPs, and MMs, every 4 wells) of a 96-well plate (CORNING, Inc.) with 100 μL of the respective culture medium. e plates were then incubated at 37°C in a humidified atmosphere of 5% CO 2 for 3 h, 1 day, 3 days, or 7 days without changing the medium. At each evaluation time point, 10 μL of the WST-8 assay reagent was added to each well and incubated at 37°C for 1 h. e plate was then gently shaken, and the absorbance of the medium (n � 6 at each point in each group) was determined using a multiplate reader at a wavelength of 450 nm. e absorbance of each medium in the vacant wells (n � 6) was also measured, and this absorbance was used as an arbitrary zero point.

e Apoptosis Assay Using Annexin V. Cryopreserved
HEKas, HDFas, ASCs, HEMa-LPs, and MMs were rapidly thawed and cultured, and cell suspensions of 1 × 10 6 cells/ml in the respective culture medium were prepared. A total of 300 μl of each cell suspension was then packed into individual plastic bags, with 16 bags prepared for each kind of cell. One bag of each cell type was preserved at room temperature without pressurization in our safety cabinet (control sample), and the other 15 were pressurized under different conditions, as mentioned above.
After pressurization, 100 μl of each cell suspension, including the nonpressurization group (control sample), was transferred to a 100 μl microtube.
en, 100 μl of Muse Annexin V & Dead Cell solution (Merck Millipore, Darmstadt, Germany) was added to each tube followed by incubation for 20 min at room temperature. e percentages of the cell population that was alive, apoptotic, or dead were analyzed using a Muse Cell Analyzer (Merck Millipore).

TEM of HDFas after Pressurization. Cryopreserved
HDFas were thawed and cultured, and a 1 mL suspension containing 1 × 10 6 HDFas was packed in a plastic bag, with 4 bags prepared in total. One bag was kept at room temperature (control sample), and the other 3 were pressurized at 190 MPa. e pressurization time was 1 s, 2 min, or 10 min. ese samples were then fixed with 2% glutaraldehyde, 0.

Statistical Analyses.
Statistical significance was assessed using the Steel Test. All data are expressed as the mean ± standard deviation. P values < 0.05 were considered to be statistically significant.

Live/Dead Cell Staining without Pressurization and after
Pressurization. Regarding the live/dead staining of HEKas, most of the cells without pressurization (control) and with HHP at any pressure for 1 s were stained by the green fluorescence derived from SYTO 10 green fluorescent nucleic acid (Figure 1(a)). e area stained by the red fluorescence derived from ethidium homodimer II nucleic acid stain then increased with HHP at any pressure for 2 min, with most of the cells stained red at 10 min (Figure 1(a)).
Most HDFas without pressurization (control) and with HHP at any pressure for 1 s were stained green (Figure 1(b)). e percentage of HDFas stained red increased with HHP at any pressure for 2 min, and most of the cells were stained red at 10 min (Figure 1(b)).
Most ASCs were stained green in the control sample and at any pressure for 1 s and 2 min. e percentage of ASCs stained red increased with HHP at any pressure for 10 min (Figure 1(c)).
Most HEMa-LPs without pressurization (control) and with HHP at any pressure for 1 s and 2 min were stained green. e percentage of HEMa-LPs stained red increased with HHP at any pressure for 10 min, and most of the cells were stained red above 170 MPa (Figure 1(d)).
Regarding the live/dead staining of MMs, most of the cells were stained green in the control sample and at any pressure for 1 s and 2 min. Most of the cells were stained red at any pressure for 10 min (Figure 1(e)).

Morphological observation of cells without pressurization and after HHP.
e micrographs of the cultured cells of the control group (without pressurization) and pressurization groups are shown in Figure 2. All cells in the control group and after HHP at any pressure for 1 s were attached and proliferated for seven days (Figures 2(a)-2(e)). HEKas after HHP at any pressure for 2 and 10 min, indicated by a red frame, were floating, which indicated that they had been inactivated (Figure 2

Results of the WST-8 Assay of Cells without Pressurization and after HHP.
e results of the objective evaluation of cell viability using the WST-8 assay are shown in Figure 3. e growth of HEKas was disturbed after HHP at any pressure for 1 s and completely inhibited after HHP for 2 and 10 min (Figure 3(a)). e growth of HDFas was disturbed after HHP at any pressure for 2 min and completely inhibited after HHP at ≥160 MPa for 10 min (Figure 3(b)). e growth of ASCs was disturbed after HHP at any pressure for 2 min and completely inhibited after HHP at any pressure for 10 min (Figure 3(c)). e growth of HEMa-LPs was disturbed after HHP at any pressure for 2 min and completely inhibited after HHP at any pressure for 10 min (Figure 3(d)). e growth of MMs was disturbed after HHP at any pressure for 2 min for 3 days, but MM cells proliferated at Day 7 ( Figure 3(e)); the growth was completely inhibited after HHP at ≥160 MPa for 10 min (Figure 3(e)).

e Apoptosis Assay of Cells Using Annexin V.
e dot plots of the apoptosis assay are shown in Figure 4. e vertical axis shows the cell viability as evaluated by the ratio of nonviable cells dyed by 7-Amino actinomycin D (7-AAD). e horizontal axis shows the ratio of the annexin V-positive cells. e lower-left quadrant of the dot plot in which both 7-AAD and Annexin V are negative shows the viable cells, and the lower-right quadrant in which 7-AAD was negative and Annexin V was positive indicates cells in the early stages of apoptosis. e upper-left quadrant in which 7-AAD was positive and Annexin V was negative indicates cells that died through pathways other than the apoptotic pathway, and the upper-right quadrant in which both 7-AAD and Annexin V were positive indicates cells that died by necrosis. Cells that were positive for both 7-AAD and Annexin V in this study were regarded as necrotic cells.
HEKas in the early stages of apoptosis were rare in each condition, and the percentage of necrotic cells increased with increasing pressure and pressurization time, with most HEKas being necrotic after HHP ≥180 MPa for 10 min   d)). e percentage of necrotic cells increased with increasing pressure, and most HDFas were necrotic after HHP at 190 MPa for 10 min (Figures 4(c) and 4 (d)). ASCs in the early stages of apoptosis were also rare, and the percentage of necrotic cells increased with increasing pressure and pressurization time, with most ASCs being necrotic after HHP ≥170 MPa for 10 min (Figures 4(e) and 4(f )). Regarding HEMa-LPs, the percentage of viable cells was larger than those of other cells; about 40% of HEMa-LPs were still alive after HHP at 190 MPa for 10 min (Figures 4(g) and 4(h)). Regarding MMs, there were a sizeable percentage of cells in the early stages of apoptosis after HHP for 10 min, and 23% of cells were in the early apoptotic phase after HHP at 190 MPa, with 69% of cells being necrotic (Figures 4(i) and 4(j)).

TEM.
e TEM findings of HDFas without HHP and after HHP at 190 MPa are shown ( Figure 5). e dendritic structure of the cell membrane was observed in the nopressurization group (Figure 5(a)) and after HHP for 1 s (Figure 5(b)). In contrast, the structure was missing after HHP for 2 min (Figure 5(c)), and the cell membrane was destroyed for 10 min (Figure 5(d)).

Discussion
e aim of this study was to explore the critical pressure and pressurization time necessary to inactivate cells related to skin or skin tumor between 150 and 200 MPa after up to 10 min of pressurization. e effect of HHP on human cells was first reported in 1961 [9], and numerous papers have been published concerning the effects of HHP on various kinds of human cells. We reported that human umbilical vein endothelial cells, human aortic smooth muscle cells, and 3T3 cells were completely killed by HHP at 200 MPa for 10 min [10], and we also showed that porcine skin [ [14].
e different types of cell death are often defined by morphological criteria [15]. In fact, we usually focus on the cell growth after culturing, and the morphologic changes in cells to determine the cell viability, as methods for detecting changes in specific enzyme, such as live/dead staining or the apoptosis assay using Annexin V, indicate cell death indirectly. In the present study, the results of the WST-8 assay after culturing and the morphology of the cultured cells showed that HHP at >150 MPa for 2 and 10 min inactivated HEKas, and HHP at >170 MPa for 10 min inactivated HDFas, ASCs and MMs, while HHP at >180 MPa for 10 min inactivated HEMa-LPs. ese results suggest that the sensitivity to HHP differs among cell types, but HHP at >180 MPa for 10 min was sufficient to inactivate all types of cells related to GCMN. is supports our previous finding that HHP at 200 MPa for 10 min was able to inactivate all cells in skin samples and GCMN. However, one limitation associated with HHP in vivo is that HHP must be used after separating the tissue from the living body, and it cannot inactivate the cells selectively and sterilize the tissue.
Regarding the cell death pathway related to HHP, Takano et al. reported that both necrosis and apoptosis were observed to be induced by pressure in human lymphoblasts [16]. It has also been reported that HHP at 100 MPa for 30 min in erythroleukemia cells and HHP at >150 MPa for 10 min in acute lymphoblastic leukemia cell lines induced apoptosis          [ 17,18], while HHP at >300 MPa for 5 min in lymphoblast-like human cells induced necrosis [8]. In addition, previous reports have shown that lymphoblast-like human cells died through apoptosis after exposure to 200 MPa for 5 min [8] and that programmed cell death was induced at 150 to 250 MPa for 5 min [19]. e cell death pathways of various cells at various pressures and for a range of pressurization durations have been discussed in detail, and whether apoptosis or necrosis, or both, occurs varies depending on the strength and duration of pressurization, as well as the cell type.
In the present study, HHP at >180 MPa for 10 min inactivated 5 kinds of cells. We considered the main death pathway of these cells to be necrosis, as the death was induced only for 10 min. e Annexin V assay detected that about 20% of MMs showed apoptosis after HHP at 190 MPa for 10 min, suggesting that some percentage of MMs died through apoptosis. However, most of the other types of cells were necrotic, according to the Annexin V assay, and TEM clearly showed the disruption of the cell membrane and swelling of the cells that indicated necrosis due to a damaged cellular membrane. e apoptotic cells detected by the Annexin V assay were negative for 7-AAD and positive for Annexin V, indicating that Annexin V was bound to the phosphatidylserine on the inner surface of the cellular membrane in those cells. is means that this assay was able to detect early apoptotic cells and necrotic cells whose membrane had been disrupted.
Previous reports have shown that the primary cells of porcine skin, human skin, and human nevus tissue were inactivated by HHP at 200 MPa for 10 min [3,5,6]. Because the cell death pathway under HHP at 190 MPa for 10 min was mainly necrosis by disruption of the cell membrane, both freshly isolated primary cells and cryopreserved cells would be able to be inactivated under the same pressure and time conditions.

Conclusions
HHP at ≥180 MPa for 10 min can inactivate various kinds of human cell related to normal skin, furthermore related to malignant skin tumor, such as MMs. ese results support our previous finding that HHP at 200 MPa for 10 min was able to inactivate all cells in skin samples and GCMN, which this pressurization condition seems to be applicable to the treatment of malignant skin tumors.

Data Availability
e data used to support the findings of this study are included in the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
NM and TY were involved in conceptualization. Data curation was performed by TM, NM, AM, and TY. Formal analysis was performed by TM, MN, and YT. NM acquired funds. TM, NM, SCN, TML, and MCN conducted investigation. NM, AM, NM, MM, HM, and TY were responsible for the methodology. Project administration was performed by NM, TY, and KK. NM was responsible for resources. Supervision was conducted by NM and KK. TM, NM, and KK were involved in validation. TM and NM were involved in visualization. TM wrote the original draft. NM and KK were involved in writing the review and editing. All authors reviewed the manuscript.