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Serum Is an Indispensable Factor in the Maintenance of the Biological Characteristics of Sweat Gland Cells

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Serum Is an Indispensable Factor in the Maintenance of the Biological Characteristics of Sweat Gland Cells MOLECULAR MEDICINE REPORTS 16: 2691-2699, 2017 Serum is an indispensable factor in the maintenance of the biological characteristics of sweat gland cells QING SUN1,2*, XIAO‑MEI DENG2*, YUN‑LIANG WANG3*, YUN‑FANG ZHEN4, FANG LI2,5, RUI‑HUA CHEN1,2, HAN‑SI LIANG1,2, FENG ZHANG2,6, MING‑DE QIN2,6 and XUE‑GUANG ZHANG1,2 1Jiangsu Institute of Clinical Immunology, The First Affiliated Hospital of Soochow University; 2The Stem Cell and Biomedical Material Key Laboratory of Jiangsu; 3Department of General Surgery, The First Affiliated Hospital of Soochow University, Soochow University, Suzhou, Jiangsu 215007; 4Institute of Pediatrics, Children's Hospital of Soochow University, Suzhou, Jiangsu 215005; 5Department of Human Anatomy, Histology and Embryology, School of Biology and Basic Medical Sciences; 6Department of Immunology, School of Biology and Basic Medical Sciences, Soochow University, Suzhou, Jiangsu 215123, P.R. China Received March 19, 2016; Accepted May 8, 2017 DOI: 10.3892/mmr.2017.6909 Abstract. The tolerance of sweat gland cells for in vitro ampli- differentiated into keratinocytes. Therefore, the SG:KGM‑2 fication and subcultivation is low as they are somatic cells. The (1:1) medium may be a suitable culture medium for HESGCs. present study aimed to formulate an optimal medium for the In conclusion, this mixed medium is a valuable compound and culture of human eccrine sweat gland cells (HESGCs) and to should be considered to be a potential supplemental medium establish a method for induction of HESGCs proliferation, for HESGCs. whilst maintaining the characteristics of sweat gland cells. HESGCs cultured in sweat gland (SG):keratinocyte growth Introduction medium‑2 (KGM‑2) (1:1) medium had a higher proliferation rate and a stable morphology compared with cells cultured in Sweat glands are major skin structures distributed across SG and KGM‑2 medium only. Reverse transcription‑quanti- human skin that are vital for temperature regulation and tative polymerase chain reaction indicated that cells cultured electrolyte balance (1,2). An increasing number of patients in the SG:KGM‑2 (1:1) medium exhibited higher expression with large area burns suffer from sweat gland defects, which levels of α‑smooth muscle actin, keratin (K)77, carcinoem- severely affect their quality of life (3). Additionally, human bryonic antigen, K8, K18, ectodysplasin A receptor, c‑Myc, eccrine sweat gland cells (HESGCs) have been reported to Kruppel‑like factor 4 and octamer‑binding transcription factor be capable of repair of wounded human skin (4,5). Therefore, 4 compared with cells cultured in SG only or KGM‑2 only increasing the availability of therapeutic sweat glands is medium. Three‑dimensional culture analysis revealed that required for future clinical studies. However, HESGCs have HESGCs cultured in SG:KGM‑2 1:1 medium differentiated a limited proliferation capacity in the currently used culture into sweat gland‑like structures, whereas cells cultured in conditions (6,7). Previous studies have determined that bone KGM‑2 only medium underwent cornification. The present marrow mesenchymal and hair follicle stem cells may be study also determined that the maintenance of the biological induced to differentiate into sweat gland cells in order to characteristics of HESGCs occurred due to the presence of fetal produce more HESGCs in vitro (8,9). However, previous bovine serum (FBS). Cells cultured in medium without FBS studies have not compared the efficacy of different basal media for the culture of sweat gland cells. Therefore, it is necessary to improve knowledge of the current culture media to be used in future studies. Correspondence to: Dr Xue‑Guang Zhang or Dr Ming‑De Qin, As presented in Table I, there are two major categories of The Stem Cell and Biomedical Material Key Laboratory of Jiangsu, media presently used to cultivate HESGCs, media with and Soochow University, 708 RenMin Road, Suzhou, Jiangsu 215007, without serum (10-18). Sweat gland (SG) medium was the P.R. China most commonly used medium, containing fetal bovine serum E‑mail: [email protected] (FBS). Similarly, keratinocyte growth medium‑2 (KGM‑2) is a E‑mail: [email protected] commonly used serum‑free medium. The present study revealed that HESGCs cultured in SG *Contributed equally medium maintained the biological characteristics of HESGCs; Key words: sweat gland cells, culture media, serum, keratinocytes, however, they demonstrated slow cell growth. HESGCs cultured tissue engineering in KGM‑2 medium demonstrated increased proliferation rates; however, the cells gradually lost the biological characteristics of sweat gland cells. The aforementioned findings of the present 2692 SUN et al: SERUM AND SWEAT GLAND CELLS study implied that competitive culture conditions are required Minneapolis, MN, USA) (19). When the sweat gland cells for HESGCs. migrated from the tissues after ~5 days in the SG medium, The present study aimed to mix the two media (SG and three different culture media [SG, KGM‑2 (Lonza Group, Ltd., KGM‑2) in order to increase cell growth and maintain the Basel, Switzerland) and SG:KGM‑2 (1:1)] were added in order biological characteristics of HESGCs. The efficiency of the to observe cellular proliferation and phenotypes. three media types was compared and the mixed medium (SG:KGM‑2 medium 1:1) was identified to provide the most RNA isolation and reverse transcription‑quantitative suitable HESGC culture conditions on the basis of prolif- polymerase chain reaction (RT‑qPCR). Total RNA was isolated eration ability and maintenance of sweat gland cell biological from the HESGCs cultured in the different media using the characteristics. Additionally, the present study identified that RNeasy Mini extraction kit (Qiagen, Inc., Valencia, CA, USA) the loss of the sweat gland cell biological characteristics in according to the manufacturer's protocol. A total of 1 µg RNA serum‑free medium occurred as the sweat gland cells rapidly was used for reverse transcription (Qiagen, Inc., Valencia, CA, differentiated into keratinocytes without serum. USA). For the qPCR assays, the primer mixes were loaded in duplicate wells in 96‑well plates and then reactions were Materials and methods performed following the addition of SYBR Green PCR Master mix (Thermo Scientific, Inc., Waltham, MA, USA) and 1 µg Human skin tissues. A total of 12 infant polydactyl skin (final) cDNA. The experiment was repeated for three times. samples were collected between January 2015 and January After pre‑denaturation at 95˚C for 5 min, 30 sec at 95˚C cooling 2016 from male children aged 1‑3 years old who had a super- to 65˚C for 45 sec for 40 cycles (Bio‑Rad Laboratories, Inc., numerary sixth finger. The samples are used for present study Hercules, CA, USA). As an internal control, GAPDH levels with the written informed consent of patients' parents and the were quantified with target genes in parallel. Normalization written approval was obtained from the ethical review board and fold‑changes were calculated using the 2‑∆ΔCq method (20), of Children's Hospital Affiliated with Soochow University and the primers used are presented in Table II. (Suzhou, China). These skin samples were used for the isola- tion of sweat gland cells. All methods were performed in Three‑dimensional culture of sweat gland cells derived accordance with the approved guidelines. All experimental from different media. Collagen I from bovine tendon (Gibco; protocols were approved by Soochow University. Copies of the Thermo Fisher Scientific, Inc.) and Matrigel (BD Biosciences, written consent provided by the subjects along with the written Franklin Lakes, NJ, USA) were used to establish a dermal approval from the review board were kept in the Children's layer on a polycarbonate membrane. The acellular collagen Hospital Affiliated with Soochow University ethical review matrix layers were allowed to stand at room temperature for board office. All experimental procedures using skin samples ~20 min and solidify, subsequently a cellular collagen matrix in the present study were reviewed and approved by the ethics layer mixed with 3.3x104 fibroblasts and 1x105 sweat gland committee. cells was added. The mixture was incubated for 1 h at 37˚C in order to allow it to solidify. When the mixtures were solidi- Isolation and culture of primary human sweat gland cells. fied, EpiLife medium (Gibco; Thermo Fisher Scientific, Inc.) Following the removal of subcutaneous tissue, the skin samples was used to cover the surface of the gels and this medium was were minced into smaller pieces with sharp scissors in phos- changed every two days. The gels were subsequently used for phate buffered saline (PBS), then epidermal and dermal tissues experiments after 21 days of culture in an incubator at 37˚C. were separated using dispase II (1 mg/ml; Roche Applied Science, Penzberg, Germany) at 4˚C for ~18 h. The dermal Sweat gland‑like structure counting standards. A protocol tissue was digested with collagenase type IV (2.5 mg/ml; to quantify the number of sweat gland‑like structures that Sigma‑Aldrich, Merck Millipore, Darmstadt, Germany) in formed in the gels was designed. A total of 3 sections were an incubator at 37˚C for 1 h. Sweat gland tissues became selected randomly with an interval of at least 100 µm to avoid accessible following digestion and were collected using a the same structure being counted repeatedly. The counting Transferpettor under an ultraviolet‑sterilized phase‑contrast standards were as follows: Sweat gland‑like structures formed inverted microscope at magnification x40. by sweat gland cells in different culture media were defined The collected sweat gland tissues were seeded into a as structures composed of 6‑15 cells in one region, which is 6‑well plate (Corning, Inc., Corning, NY, USA) in SG medium similar to the transverse section of normal sweat gland tissue. at a density of ~120 tissues/well. The SG medium contained The number of structures was confirmed under an optical Dulbecco's modified Eagle's medium‑F12 (DMEM‑F12, microscope (magnification, x10 and x40).
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