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An improved method of culturing somatotropic cells from rat adenohypophysis
Tissue and Cell 58 (2019) 93–98
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An improved method of culturing somatotropic cells from rat adenohypophysis
T ⁎
Jian Maoa,1, Yun Baoa,1, Fen Meia, Xixian Liaob, Fan Liua, Lizhi Zhouc, Songtao Qia, , ⁎ Binghui Qiua, a
Department of Neurosurgery, Nanfang Hospital, Southern Medical University, Guangzhou, China The First School of Clinical Medicine, Southern Medical University, Guangzhou, China c Department of Biostatistics, School of biostatistics, Southern Medical University, Guangzhou, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Pericytes Improved method Primary culture Somatotropic cells
This study aimed to propose a simple and practical method for culturing primary rat somatotropic cells in vitro free of pericytes contamination. Rat adenohypophyses were randomly divided into two groups. An improved method was used in group A (digesting adenohypophysis with 0.25% trypsin-EDTA, followed by removing pericytes by double filtration and using serum-free medium for culturing somatotropic cells). The traditional method was used in group B (digesting adenohypophysis with 0.35% collagenase, using serum medium for culturing somatotropic cells, and removing pericytes by changing the culture dish). The numbers and viability of somatotropic cells were higher in group A than in group B after 6 days. GH secretion of somatotropic cells was also higher in group A than in group B. Besides, the pericytes grew rapidly only in group B after 3 days. α-SMA, type I collagen, and type III collagen had weaker expression in group A. Also, the viability of pericytes decreased in group A. The improved method could solve the problem of pericytes contamination, and the culture of primary rat somatotropic cells in vitro was successful. This method can be used for other primary cultures with pericytes contamination.
1. Introduction Growth hormone (GH) is an important hormone produced by the anterior lobe of the pituitary gland (Brinkman et al., 2018; Veldhuis, 2008; Yuce et al., 2018). It promotes linear growth in children. It is also a pivotal anabolic hormone with stimulatory effects on protein synthesis (especially in the liver, spleen, kidney, and red blood cells) and lipolysis (Brinkman et al., 2018; Corpas et al., 1993; Ranke and Wit, 2018). Age or inflammation-related GH hyposecretion appears to be strongly related to reduced survival (Bartke and Darcy, 2017; Spadaro et al., 2016). Investigations have shown that somatotropic cells located in the adenohypophysis secrete GH. However, no cell line of somatotropic cells is available for research currently. Therefore, primary cultures of somatotropic cells are important to examine the role of GH in physiological regulation. Body tissues have many stromal cells besides functional cells, especially fibroblasts (McAnulty, 2007). After cell digestion, the suspension often gets mixed with a large number of fibroblasts, leading to cell contamination. The somatotropic cells are mature glandular
epithelial cells which represent a large proportion of endocrine cells dissociated from the adenohypophysis (Grandi et al., 2017). Somatotropic cells are present in suspension culture and frequently get contaminated with pericytes, which also named perivascular fibroblasts (Bijkerk et al., 2019). The somatotropic cells are more likely to die in subsequent cultures because they are difficult to culture in vitro, thereby restricting a better understanding of the role of somatotropic cells in growth and disease. Traditional methods involve the digestion of adenohypophysis with 0.35% collagenase and use of DMEM/F12 media supplemented with fetal bovine serum (FBS) to culture endocrine cells in the pituitary glands (Balen et al., 1995; Rodriguez-Pacheco et al., 2013; Vale et al., 1972). FBS has the tendency to stimulate pericytes and induce morphological changes in epithelial cells giving them a fibroblasts-like shape. Therefore, culture dishes need to be changed multiple times for removing pericytes. However, frequent changes lead to the loss of a number of somatotropic cells, while a few changes cannot solve the problem of pericytes contamination. This may be related to the serum medium used in traditional methods, which has a strong stimulatory
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Corresponding authors at: Department of Neurosurgery, Nanfang Hospital, Southern Medical University, Guangzhou Dadao Bei Street 1838#, Guangzhou, China. E-mail addresses: [email protected] (S. Qi), [email protected] (B. Qiu). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.tice.2019.04.011 Received 2 April 2019; Received in revised form 27 April 2019; Accepted 29 April 2019 Available online 30 April 2019 0040-8166/ © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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They were incubated at 37 °C in a 5% CO2 atmosphere, and the medium was replaced with serum-free medium (DMEM/F12 containing 0.5% Bovine Serum Albumin, B27, and N2 supplements besides 20 ng/mL bFGF and EGF) and 1% antibiotic–antimycotic solution after 24 h of culture.
effect on pericytes (Shahdadfar et al., 2005). Therefore, a method for culturing somatotropic cells free of pericytes contamination needs to be established urgently. For further understanding the biological characteristics of somatotropic cells, this study devotes to propose a simple and practical method for culturing primary rat somatotropic cells in vitro which can be free of pericytes contamination.
2.5. Preparation of a single-cell suspension of somatotropic cells using the traditional method
2. Materials and methods The suspension was prepared using the method described in previous studies (Balen et al., 1995; Rodriguez-Pacheco et al., 2013; Vale et al., 1972). Adenohypophyses were dispersed in 0.35% collagenase for 30 min at 37 °C. Consequently, the cells were filtered through the 70-μm cell strainer. Approximately 3.0 × 105 cells were obtained from each lobe. The cellular viability, as estimated using the trypan blue test, usually exceeded 90%. The cells were washed three times with PBS containing the antibiotics and antimycotics. Finally, the dispersed somatotropic cells were plated at a density of 3.0 × 105 onto 24-well culture plates in 1 mL of serum medium and 1% antibiotic–antimycotic solution. They were incubated at 37 °C in a 5% CO2 atmosphere. The plate needed to be changed after the number of pericytes exceeds 50%.
2.1. Animal housing and procedure 60 male Sprague–Dawley (SD) rats (weighing 200 ± 10 g; laboratory animal center, Southern Medical University) having access to standard chow and water were housed under standard laboratory conditions with a 12-h light–dark cycle at room temperature (24 °C ± 2 °C) during the experimental period. All procedures conformed to the regulations of Southern Medical University institutional animal care and the ARRIVE guidelines and were performed in accordance with the UK Animals (Scientific Procedures) Act, 1986, and associated guidelines; EU Directive 2010/63/EU for animal experiments; or the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978). The study was approved by the Nanfang Hospital animal ethics committee (ID: NFYY-2016-107). In addition, every effort was made to minimize the number of animals used in the study and their sufferings.
2.6. Culture of pericytes After removing the neurointermediate lobe, anterior pituitary lobes from SD rats were dissociated into single cells using successive incubations in 0.25% Trypsin-EDTA. For pericytes, freshly dissociated cells were cultured in DMEM, containing 10% FBS at 37 °C and 5% CO2 waiting for pericytes crawling out. The pericytes were incubated with serum or serum-free medium for subsequent experiments. The medium was changed every 2–3 days. Positive identifcation of the cells was achieved by positive staining for desmin.
2.2. Antibodies The following antibodies were used in the study: desmin rabbit polyclonal antibody (ab32362; Abcam, Cambridge, UK), GH rabbit monoclonal antibody (ab134078, Abcam, Cambridge, England), α-SMA rabbit polyclonal antibody (ab5694, Abcam, Cambridge, England), 4,6diamidino-2-phenylindole (DAPI) (CAS 28718-90-3, Santa Cruz Biotechnology, Dallas, Texas, USA), type I collagen rabbit polyclonal antibody (abs131984, Absin, Shanghai, China), type III collagen rabbit polyclonal antibody (abs131560, Absin, Shanghai, China), Alexa Fluor 488 and 555-conjugated streptavidin secondary antibodies (Life Technologies, Carlsbad, California, USA), HRP goat anti-rabbit IgG (AS014, ABclonal, Wuhan, China), and GAPDH mouse monoclonal antibody (WHO79859, ABclonal, Wuhan, China).
2.7. Immunofluorescence The cells were seeded on Matrigel and poly-L-lysine-coated 12-mm glass coverslips in 24-well plates and allowed to attach for 5 days at 37 °C. They were then fixed in 4% paraformaldehyde (PFA) in PBS for 15 min at room temperature. They were permeabilized using 0.5% Triton X-100 (Solarbio, Beijing, China) in PBS for 10 min and blocked for 1 h with 5% BSA in PBS (both steps were done at room temperature). They were subsequently immunostained at 4 °C overnight with rabbit anti-rat desmin polyclonal antibody (1: 50 dilution) or rabbit anti-rat GH monoclonal antibody (1: 200 dilution) diluted in blocking buffer, followed by the incubation with secondary antibodies conjugated to Alexa Fluor 488 and 555-conjugated streptavidin secondary antibodies (1:500 dilution) for 1 h at room temperature. The coverslips were then washed with PBS and mounted on microscope slides with ProLong Gold Antifade Reagent containing DAPI to counterstain the nuclei. Samples were examined with a Leica DMI3000 B microscope.
2.3. Sampling SD rats were anesthetized with inhalation of isofluorane (5% induction; 2–3% maintenance) and euthanized by cervical dislocation. The adenohypophyses were dissected, mixed with phosphate-buffered saline (PBS), and then transferred to Biological Safety Cabinet rapidly. After dissecting, the adenohypophyses were randomly and equally divided into group A (improved method) and group B (traditional method).
2.8. Cell counting 2.4. Preparation of a single-cell suspension of somatotropic cells using the improved method
The images of somatotropic cells in group A or group B were taken using an inverted microscope (Leica DMI3000 B, Wetzlall, Germany) on days 1, 3, and 6. The somatotropic cells in images were analyzed using a TissueFAXS Plus flow-type tissue quantitative analyzer (TissueGnostics GmbH, Vienna, Austria).
Adenohypophyses were cut into pieces using a sterile tweezer and dispersed in 0.25% trypsin-EDTA (Gibco BRL, MD, USA) for 30 min at 37 °C. The cells were filtered through a 70-μm cell strainer first and then a 20-μm filter. Approximately 5.0 × 105 cells were usually obtained from each lobe. The cellular viability, as estimated using the trypan blue test, usually exceeded 90%. The cells were washed three times with PBS containing the antibiotics and antimycotics (Gibco BRL). Finally, the dispersed somatotropic cells were plated at a density of 3.0 × 105 onto 24-well culture plates in 1 mL of serum medium (DMEM/F12; Gibco BRL) supplemented with 10% FBS (Gemini BioProducts, California, USA) and 1% antibiotic–antimycotic solution.
2.9. Cell counting kit-8 analysis Cell counting kit-8 assay (CCK8; Dojindo, Kumamoto, Japan) was used to determine cell proliferation. Briefly, 60,000 cells per well were seeded into 96-well plates coated with poly-L-lysine. A group without cells served as the blank. Subsequently, 10 μL of CCK8 solution was added to cells in each well at different time points, followed by 94
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well of a Costar Transwell chamber (8 μM; Corning Life Sciences, MA, USA) in DMEM/F12. The latter were seeded in serum or serum-free medium, which was replaced with 10% FBS-containing medium after 24 h. The cells that had migrated to the bottom side of the membrane were fixed in 70% ethanol and stained with Giemsa stain solution 24 h after plating. Subsequently, the non-migrating cells in the upper chamber were removed using a cotton-tipped applicator. The membranes were mounted onto object slides, and six random fields per slide were counted with a 10× or 20× lens objective. At least three independent experiments were performed for each assay.
incubation at 37 °C for 1 h. The absorbance was measured at 450 nm on a microplate reader. The difference between the optical density (OD) of cells in the medium and that of blank (medium-only) wells is proportional with the survival/proliferation of cells. A curve was plotted for the change in cell activity with serum and serum-free media, using time as the x-axis and OD as the y-axis. 2.10. Enzyme-linked immunosorbent assay GH secretions of somatotropic cells cultured in group A and group B were compared using enzyme-linked immunosorbent assay (ELISA). Briefly, the cells were cultured in 24-well plates from day 1 to day 6. The cell supernatant was collected after each time point and kept at room temperature for 30 min, followed by centrifugation at 1000 rpm for 20 min. For fulfilling the criteria of ELISA kit (EZRMGH-45 K, Millipore, Burlington, Massachusetts, USA), the supernatant was concentrated using Pierce Protein Concentrator, 5 K (88534, Thermo Scientific, Waltham, Massachusetts, USA), following which GH in the supernatant was used immediately for ELISA analyses or, if not, aliquoted and stored at −80 °C for subsequent use. The samples were diluted and subjected to standard ELISA assays to determine GH levels in accordance with the manufacturer`s protocol.
2.14. Statistical analysis All experiments were run in triplicate. The results were expressed as mean ± standard deviation (SD). Data between groups were compared using the Student t test. Statistical analysis was conducted using SPSS 20.0 (IBM Corp., NY, USA). P values less than 0.05 were considered statistically significant. 3. Results 3.1. Comparison of the numbers of somatotropic cells in group A and group B
2.11. Western blot analysis Individual cells were released from the isolated adenohypophyses using trypsin/collagenase treatment. To compare the differences in the number of somatotropic cells between group A and group B with the extension of time, the microscopic images of cells were taken after 1 day, 3 days, and 6 days. As showed in Fig. 1A, spindle cells grew up only in group B on day 3. Then, immunofluorescence was performed to identify the two kinds of cells in group A and group B on day 3. GH was expressed only in group A, indicating that the cells in group A were pure somatotropic cells. But both desmin and GH were expressed in group B, indicating that the cells in group B were somatotropic cells mixed with pericytes (Fig. 1B). Therefore, the cell suspension was transferred in another plate for removing pericytes in group B. Next, the somatotropic cells in group A and group B were counted after 1 day, 3 days, and 6 days. The numbers of somatotropic cells in group A were almost constant after 6 days but decreased obviously in group B. On day 6, significantly more cells were found in group A than in group B (Fig. 1C; P < 0.01). Thus, compared with the traditional method, the improved method was more efficient for culturing primary somatotropic cells.
The cells were harvested, lysed in RIPA buffer (Thermo Scientific), and supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific). Protein extracts were subjected to centrifugation (12,000g) at 4 °C, and protein concentrations were determined using the BCA assay (Pierce). Protein was separated on either NativePAGE Bis-Tris Gel System or 4%–12% SDS-Tris-Glycine Protein Gel. Separated proteins were transferred onto polyvinylidene difluoride membranes (Millipore). The membranes were blocked with 5% nonfat dry milk in TBS and incubated with rabbit anti-rat α-SMA polyclonal antibody (1:100 dilution), rabbit anti-rat type III collagen polyclonal antibody (1:500 dilution) or rabbit anti-rat type I collagen polyclonal antibody (1:500 dilution). Subsequently, the membranes were washed and incubated with appropriate secondary, HRP goat anti-rabbit IgG (1:1000 dilution). Proteins were visualized using autoradiography and enhanced chemiluminescence (ECL; Amersham Biosciences, GE Healthcare). 2.12. Scratch wound healing assay
3.2. Comparison of the status of pericytes in serum-free and serum media
The migration of pericytes in serum or serum-free medium was assessed using the scratch wound healing assay proposed by Rodriguez et al (Rodriguez et al., 2005). Briefly, the cells (3 × 105) were seeded into a six-well plate in 2 mL of serum medium and antibiotics. The cells were incubated at 37 °C and in a 5% CO2 atmosphere until confluence. Once confluent, monolayer cells in each dish were subjected to a mechanical wound by scratching with a 200-μL pipette tip, parallel to the grid bars along the central grid line. Wound placement was checked with a Leica microscope (Leica DMI3000 B). The medium was removed and the cells were washed five times with PBS, followed by the serum or serum-free treatments. The cells along the induced wound in each dish were taken after 0 and 24 h using a digital camera (Olympus Camedia 34–40 zoom, 5× magnification) attached to a light microscope. Computer program Image J (National Institutes of Health, MD, USA) was used to measure the area of initial damage (images taken at time 0) and the remaining damage after 24 h. The percentage of wound repair was calculated.
Next, the study investigated the established effect of the serum and serum-free medium on pericytes because pericytes grew up only in group B. Firstly, these cells obtained from adenohypophysis specimens were identified as pericytes by positive staining for desmin (Fig. 2A) (Tsukada et al., 2016). The results of Western blot analysis indicated that compared with the serum medium, the expression of ɑ-SMA (a marker of activating pericytes), type I collagen, and type III collagen was poor in the serum-free medium (Fig. 2B). The proliferation of pericytes was examined using CCK8 analysis. The OD of pericytes was significantly lower in the serum-free medium than in the serum medium after 24 h which indicated a lower proliferative activity of pericytes in the serum-free medium (Fig. 2C; P < 0.001). The study then investigated whether the modulation of the serum-free medium affected the migration of pericytes. None of the pericytes passed through the chamber in the serum-free medium in Boyden chamber assays (Fig. 2D). Similarly, as shown in Fig. 2E and F, the ratio of scratch wound healing was lower when pericytes were cultured in the serum-free medium compared with the serum medium (P < 0.001). These findings indicated that compared with the serum medium, the serum-free medium did not support the survival of pericytes.
2.13. Migration assay The cell migration was assessed using a Boyden chamber assay. For these experiments, 1 × 105 cells (pericytes) were seeded onto the upper 95
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Fig. 1. Differences in the numbers of somatotropic cells between group A and group B at different time points. A. Microscopic images of somatotropic cells were taken on days 1, 3, and 6. Spindle cells grew up in group B on the third day. The medium was changed for wiping off spindle cells in group B. Red arrow: somatotropic cells. Green arrow: spindle cells. Scale bar = 100 μm. B. Cells in group A and group B on the third day were identified by immunofluorescence. The cells in group A only expressed GH (red), indicating that the cells in group A were pure somatotropic cells. The cells in group B expressed GH (red) and desmin (green), indicating that the cells in group B were somatotropic cells mixed with pericytes. DAPI (blue stain) was used as a nuclear marker. Scale bar = 20 μm. C. A representative graph for cell counts of somatotropic cells in group A and group B (n = 3/group) on days 1, 3, and 6. Data are shown as mean ± SD. *P < 0.05; **P < 0.01.
Fig. 2. Comparison of the growth status of pericytes in the serum and serum-free media for 24 h. A. Identification of primary pericytes in culture. Pericytes are characterized by positive staining for desmin (green). DAPI (blue stain) was used as a nuclear marker. Scale bar = 20 μm. B. Western blot analysis was used to detect the expression of ɑ-SMA, type I collagen, and type III collagen in pericytes. GAPDH expression was monitored to confirm equal protein loading and blotting. C. Detecting the viability of pericytes cultured in the serum and serum-free media using CCK8 assay (n = 3/group). Data are shown as mean ± SD. **P < 0.01; ***P < 0.001. D. Cell migration analysis of pericytes cultured in serum and serum-free media as evaluated using Boyden chamber assay. Scale bar = 100 μm. E. Scratch wound healing assay of pericytes after indicated treatments for 24 h. Scale bar = 100 μm. F. Statistical analyses of the scratch wound area of pericytes are shown (n = 3/group). Data are shown as mean ± SD. ***P < 0.001. 96
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Fig. 3. Comparison of the status of somatotropic cells in group A and group B within 6 days. A. CCK8 assay was used to detect the viability of somatotropic cells in group A and group B (n = 3/group). Data are shown as mean ± SD. **P < 0.01; ***P < 0.001. B. ELISA assay was used to analyze the GH secretion in somatotropic cell supernatant in group A and group B (n = 3/group). Data are shown as mean ± SD. **P < 0.01; ***P < 0.001.
treatments had much higher cell isolation rates than collagenase treatment, with no damage to somatotropic cells. Double filtration was used for removing pericytes, which decreased the number of pericytes at the beginning of the culture. Moreover, the components in the culture medium (DMEM/F12 containing 0.5% BSA, B27, and N2 supplements besides 20 ng/mL bFGF and EGF) were optimized and the presence of somatotropic cells was confirmed. The main obstacle to establishing pure somatotropic cells in vitro is pericytes contamination of the cultures. In the present study, somatotropic cells in the adenohypophysis were isolated by two-step filtration and cultured in the serum-free medium. The traditional method usually involves filtering the harvested tissues using a 70-μm cell strainer for obtaining the cell suspension. However, it cannot separate the pericytes from somatotropic cells. The diameters of pericytes and somatotropic cells are different. After many experiments, it was found that somatotropic cells could pass through a 20-μm cell strainer, whereas pericytes could not. The number of pericytes was minimized when a single-cell suspension was obtained by two-step filtration. FBS is the supplement most widely used for cell culture. It provides hormones, proteins, minerals, and several other factors (Paula et al., 2015). The pericytes show superior growth and gradually replace somatotropic cells in the serum medium. The mode of separation used in the traditional method is not preferred because it is associated with the loss of somatotropic cells. The main reason is that FBS strongly supports the growth of pericytes, as reported earlier (Shahdadfar et al., 2005). Therefore, finding the substitutes of FBS is essential to ensure cell manipulation. Developing a chemically defined cell culture condition that supports the growth of somatotropic cells but not of pericytes is an important issue. Different studies have attempted to replace FBS with human-derived alternatives (Aldahmash et al., 2011; Bernardo et al., 2007; Bieback et al., 2009; Kocaoemer et al., 2007; Lindroos et al., 2010; Shahdadfar et al., 2005) or serum-free defined media (Agata et al., 2009; Chase et al., 2012; Mimura et al., 2011; Rodrigues et al., 2010; Tarle et al., 2011; van der Valk et al., 2010). Therefore, finding a medium formula suitable for somatotropic cells but not for pericytes is possible. The serum-free defined media comprise synthetic supplements that replace serum and reduce variability, thus offering the most promising alternative. Thus, this study proposed the use of N2/B27/bFGF/EGF, which was the regular supplement for culturing the primary cells in the serum-free medium (Chen et al., 2016; Fauquier et al., 2008). The culture of primary somatotropic cells using the medium with N2/B27/bFGF/EGF was successful, and the morphologic characteristics of somatotropic cells cultured in serum-free medium were comparable to those of cells in FBS. The somatotropic cells in group A were rounded, bright, and floating; they formed several colonies. However, the somatotropic cells in group B had a pericytes-like shape, reduced brightness, and tendency to cluster at the confluence. These observations agreed with another published study that revealed similar
3.3. Comparison of the status of somatotropic cells in group A and group B To assess the status of somatotropic cells in group A and group B, CCK8 assay was performed to detect the viability of somatotropic cells in the two groups. 60,000 cells were plated per well from the two groups. The result indicated that the viability of somatotropic cells in group A was quite stable within 6 days, but it decreased in group B (Fig. 3A; P < 0.001). Next, ELISA assay was performed to detect GH secretion of somatotropic cells in group A and group B. The same number cells were cultured in plates from day 1 to day 6. And then the cell supernatant was collected. The result showed that the GH secretion of somatotropic cells in group B increased within 3 days and then remained constant, indicating that somatotropic cells in group B could not secrete GH after 3 days. On the contrary, the GH secretion of somatotropic cells in group A increased within 6 days, indicating that somatotropic cells in group A secreted GH continuously. After 6 days, the GH secretion of somatotropic cells was significantly higher in group A than in group B (Fig. 3B; P < 0.001). These results collectively indicated that somatotropic cells in group A were in a better status compared with group B. 4. Discussion The mechanisms underlying adenohypophysis and neuroendocrine regulation have gained immense attention in recent years. Many immortalized cell lines that secrete various kinds of hormones have been developed to meet the research needs (Ooi et al., 2004). However, the transformed immortalized cell lines generally grow well under in vitro culture conditions, thus exhibiting limited differentiation properties compared with their wild-type counterparts. Also, the pathophysiological mechanism may differ from the mechanism for primary cultured cells. Therefore, the use of primary cultured cells is important for studying their differentiation properties (Ooi et al., 2004). Nevertheless, somatotropic cells are not capable of propagating and pericytes contamination in primary culture is a common problem (Kassen et al., 1996; Vidrich et al., 1988). Therefore, culturing primary somatotropic cells without pericytes contamination needs further exploring. This study aimed to propose a method for culturing somatotropic cells. The use of the improved and traditional methods for isolating and culturing somatotropic cells in vitro was evaluated. In the present study, an improved method was developed to isolate somatotropic cells, and the culturing conditions required for the survival of somatotropic cells were determined. The adenohypophysis was chosen rather than the entire pituitary gland because somatotropic cells are located in the adenohypophysis (Gajkowska et al., 2006), thereby providing sufficient pure cells. In addition, both enzyme digestion and tissue culture methods were used to isolate primary somatotropic cells, hence greatly improving the rate of obtaining primary somatotropic cells. The previous method was modified by cutting the adenohypophysis into pieces and digesting it with 0.25% trypsin-EDTA because trypsin-EDTA 97
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characteristics (Kokubu et al., 2015). Further, differences in the survival rates of somatotropic cells were observed under different culture conditions. The results indicated that the culture conditions had a marked effect after 3 days; the serum-free medium provided a better survival rate for somatotropic cells. Conversely, the viability of pericytes in the serum-free medium used in group A, compared with the serum medium, was significantly inhibited. Therefore, these methods could solve the problem of pericytes contamination successfully.
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Shahdadfar, A., Fronsdal, K., Haug, T., Reinholt, F.P., Brinchmann, J.E., 2005. In vitro expansion of human mesenchymal stem cells: choice of serum is a determinant of cell proliferation, differentiation, gene expression, and transcriptome stability. Stem Cells 23 (9), 1357–1366. Spadaro, O., Goldberg, E.L., Camell, C.D., Youm, Y.H., Kopchick, J.J., Nguyen, K.Y., Bartke, A., Sun, L.Y., Dixit, V.D., 2016. Growth hormone receptor deficiency protects against age-related NLRP3 inflammasome activation and immune senescence. Cell Rep. 14 (7), 1571–1580. Tarle, S.A., Shi, S., Kaigler, D., 2011. Development of a serum-free system to expand dental-derived stem cells: PDLSCs and SHEDs. J. Cell. Physiol. 226 (1), 66–73. Tsukada, T., Azuma, M., Horiguchi, K., Fujiwara, K., Kouki, T., Kikuchi, M., Yashiro, T., 2016. Folliculostellate cell interacts with pericyte via TGFbeta2 in rat anterior pituitary. J. Endocrinol. 229 (2), 159–170. Vale, W., Grant, G., Amoss, M., Blackwell, R., Guillemin, R., 1972. Culture of enzymatically dispersed pituitary cells: functional validation of a method. Endocrinology 91 (2), 562–572. van der Valk, J., Brunner, D., De Smet, K., Fex Svenningsen, A., Honegger, P., Knudsen, L.E., Lindl, T., Noraberg, J., Price, A., Scarino, M.L., Gstraunthaler, G., 2010. Optimization of chemically defined cell culture media–replacing fetal bovine serum in mammalian in vitro methods. Toxicol. In Vitro 24 (4), 1053–1063. Veldhuis, J.D., 2008. Aging and hormones of the hypothalamo-pituitary axis: gonadotropic axis in men and somatotropic axes in men and women. Ageing Res. Rev. 7 (3), 189–208. Vidrich, A., Ravindranath, R., Farsi, K., Targan, S., 1988. A method for the rapid establishment of normal adult mammalian colonic epithelial cell cultures. In Vitro Cell. Dev. Biol. 24 (3), 188–194. Yuce, O., Yalcin, N.G., Bideci, A., Doger, E., Emeksiz, H.C., Hasanreisoglu, M., Aktas, Z., Camurdan, O., Cinaz, P., 2018. Retinal neural and vascular structure in isolated growth hormone deficiency children and evaluation of growth hormone treatment effect. J. Clin. Res. Pediatr. Endocrinol. 10 (2), 113–118.
5. Conclusion This study proposed a more rational method for obtaining single, intact primary somatotropic cells that were of good quality in terms of survival rate, integrity, and general morphology. The improved method helped avoid pericytes contamination. The primary somatotropic cells were not readily stimulated and the risk of contamination was minimized because the cells needed no purification again in subsequent cultures. The findings might provide a solid foundation for studying the biological characteristics of GH. This method can be refer to other studies on primary cultures contaminated with pericytes. Funding This study was supported by National Science and Technology Infrastructure Program (2014BAI04B01), Scienceand Technology Planning Project of Guangdong Province, China (2016A020213006) (2017A030303021), Chinese National Natural Science Foundation (81701197), Natural Science Foundation of Guangdong Province (2017A030310111), President Foundation of Nanfang Hospital, Southern Medical University (2017B009), Clinical Research Startup Program of Southern Medical University by High-level University Construction Funding of Guangdong Provincial Department of Education (LC2016PY012). Authors contributions Jian Mao, Yun Bao, Fen Mei performed the experiments. Xixian Liao, Fan Liu wrote the paper. Lizhi Zhou performed data analysis. Songtao Qi, Binghui Qiu conceived the ideas and designed the experiments Conflict of interest The authors declare that they have no conflict of interests. References Agata, H., Watanabe, N., Ishii, Y., Kubo, N., Ohshima, S., Yamazaki, M., Tojo, A., Kagami, H., 2009. Feasibility and efficacy of bone tissue engineering using human bone marrow stromal cells cultivated in serum-free conditions. Biochem. Biophys. Res. Commun. 382 (2), 353–358. Aldahmash, A., Haack-Sorensen, M., Al-Nbaheen, M., Harkness, L., Abdallah, B.M., Kassem, M., 2011. Human serum is as efficient as fetal bovine serum in supporting proliferation and differentiation of human multipotent stromal (mesenchymal) stem cells in vitro and in vivo. Stem Cell Rev. 7 (4), 860–868. Balen, A.H., Er, J., Rafferty, B., Rose, M., 1995. Characterization of a rat anterior pituitary cell bioassay. In Vitro Cell. Dev. Biol. Anim. 31 (4), 316–322. Bartke, A., Darcy, J., 2017. GH and ageing: pitfalls and new insights. Best Pract. Res. Clin. Endocrinol. Metab. 31 (1), 113–125. Bernardo, M.E., Avanzini, M.A., Perotti, C., Cometa, A.M., Moretta, A., Lenta, E., Del Fante, C., Novara, F., de Silvestri, A., Amendola, G., Zuffardi, O., Maccario, R., Locatelli, F., 2007. Optimization of in vitro expansion of human multipotent mesenchymal stromal cells for cell-therapy approaches: further insights in the search for a fetal calf serum substitute. J. Cell. Physiol. 211 (1), 121–130. Bieback, K., Hecker, A., Kocaomer, A., Lannert, H., Schallmoser, K., Strunk, D., Kluter, H., 2009. Human alternatives to fetal bovine serum for the expansion of mesenchymal stromal cells from bone marrow. Stem Cells 27 (9), 2331–2341. Bijkerk, R., Au, Y.W., Stam, W., Duijs, J., Koudijs, A., Lievers, E., Rabelink, T.J., van Zonneveld, A.J., 2019. Long non-coding RNAs Rian and miat mediate myofibroblast formation in kidney fibrosis. Front. Pharmacol. 10, 215. Chase, L.G., Yang, S., Zachar, V., Yang, Z., Lakshmipathy, U., Bradford, J., Boucher, S.E.,
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An improved method of culturing somatotropic cells from rat adenohypophysis
T ⁎
Jian Maoa,1, Yun Baoa,1, Fen Meia, Xixian Liaob, Fan Liua, Lizhi Zhouc, Songtao Qia, , ⁎ Binghui Qiua, a
Department of Neurosurgery, Nanfang Hospital, Southern Medical University, Guangzhou, China The First School of Clinical Medicine, Southern Medical University, Guangzhou, China c Department of Biostatistics, School of biostatistics, Southern Medical University, Guangzhou, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Pericytes Improved method Primary culture Somatotropic cells
This study aimed to propose a simple and practical method for culturing primary rat somatotropic cells in vitro free of pericytes contamination. Rat adenohypophyses were randomly divided into two groups. An improved method was used in group A (digesting adenohypophysis with 0.25% trypsin-EDTA, followed by removing pericytes by double filtration and using serum-free medium for culturing somatotropic cells). The traditional method was used in group B (digesting adenohypophysis with 0.35% collagenase, using serum medium for culturing somatotropic cells, and removing pericytes by changing the culture dish). The numbers and viability of somatotropic cells were higher in group A than in group B after 6 days. GH secretion of somatotropic cells was also higher in group A than in group B. Besides, the pericytes grew rapidly only in group B after 3 days. α-SMA, type I collagen, and type III collagen had weaker expression in group A. Also, the viability of pericytes decreased in group A. The improved method could solve the problem of pericytes contamination, and the culture of primary rat somatotropic cells in vitro was successful. This method can be used for other primary cultures with pericytes contamination.
1. Introduction Growth hormone (GH) is an important hormone produced by the anterior lobe of the pituitary gland (Brinkman et al., 2018; Veldhuis, 2008; Yuce et al., 2018). It promotes linear growth in children. It is also a pivotal anabolic hormone with stimulatory effects on protein synthesis (especially in the liver, spleen, kidney, and red blood cells) and lipolysis (Brinkman et al., 2018; Corpas et al., 1993; Ranke and Wit, 2018). Age or inflammation-related GH hyposecretion appears to be strongly related to reduced survival (Bartke and Darcy, 2017; Spadaro et al., 2016). Investigations have shown that somatotropic cells located in the adenohypophysis secrete GH. However, no cell line of somatotropic cells is available for research currently. Therefore, primary cultures of somatotropic cells are important to examine the role of GH in physiological regulation. Body tissues have many stromal cells besides functional cells, especially fibroblasts (McAnulty, 2007). After cell digestion, the suspension often gets mixed with a large number of fibroblasts, leading to cell contamination. The somatotropic cells are mature glandular
epithelial cells which represent a large proportion of endocrine cells dissociated from the adenohypophysis (Grandi et al., 2017). Somatotropic cells are present in suspension culture and frequently get contaminated with pericytes, which also named perivascular fibroblasts (Bijkerk et al., 2019). The somatotropic cells are more likely to die in subsequent cultures because they are difficult to culture in vitro, thereby restricting a better understanding of the role of somatotropic cells in growth and disease. Traditional methods involve the digestion of adenohypophysis with 0.35% collagenase and use of DMEM/F12 media supplemented with fetal bovine serum (FBS) to culture endocrine cells in the pituitary glands (Balen et al., 1995; Rodriguez-Pacheco et al., 2013; Vale et al., 1972). FBS has the tendency to stimulate pericytes and induce morphological changes in epithelial cells giving them a fibroblasts-like shape. Therefore, culture dishes need to be changed multiple times for removing pericytes. However, frequent changes lead to the loss of a number of somatotropic cells, while a few changes cannot solve the problem of pericytes contamination. This may be related to the serum medium used in traditional methods, which has a strong stimulatory
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Corresponding authors at: Department of Neurosurgery, Nanfang Hospital, Southern Medical University, Guangzhou Dadao Bei Street 1838#, Guangzhou, China. E-mail addresses: [email protected] (S. Qi), [email protected] (B. Qiu). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.tice.2019.04.011 Received 2 April 2019; Received in revised form 27 April 2019; Accepted 29 April 2019 Available online 30 April 2019 0040-8166/ © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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They were incubated at 37 °C in a 5% CO2 atmosphere, and the medium was replaced with serum-free medium (DMEM/F12 containing 0.5% Bovine Serum Albumin, B27, and N2 supplements besides 20 ng/mL bFGF and EGF) and 1% antibiotic–antimycotic solution after 24 h of culture.
effect on pericytes (Shahdadfar et al., 2005). Therefore, a method for culturing somatotropic cells free of pericytes contamination needs to be established urgently. For further understanding the biological characteristics of somatotropic cells, this study devotes to propose a simple and practical method for culturing primary rat somatotropic cells in vitro which can be free of pericytes contamination.
2.5. Preparation of a single-cell suspension of somatotropic cells using the traditional method
2. Materials and methods The suspension was prepared using the method described in previous studies (Balen et al., 1995; Rodriguez-Pacheco et al., 2013; Vale et al., 1972). Adenohypophyses were dispersed in 0.35% collagenase for 30 min at 37 °C. Consequently, the cells were filtered through the 70-μm cell strainer. Approximately 3.0 × 105 cells were obtained from each lobe. The cellular viability, as estimated using the trypan blue test, usually exceeded 90%. The cells were washed three times with PBS containing the antibiotics and antimycotics. Finally, the dispersed somatotropic cells were plated at a density of 3.0 × 105 onto 24-well culture plates in 1 mL of serum medium and 1% antibiotic–antimycotic solution. They were incubated at 37 °C in a 5% CO2 atmosphere. The plate needed to be changed after the number of pericytes exceeds 50%.
2.1. Animal housing and procedure 60 male Sprague–Dawley (SD) rats (weighing 200 ± 10 g; laboratory animal center, Southern Medical University) having access to standard chow and water were housed under standard laboratory conditions with a 12-h light–dark cycle at room temperature (24 °C ± 2 °C) during the experimental period. All procedures conformed to the regulations of Southern Medical University institutional animal care and the ARRIVE guidelines and were performed in accordance with the UK Animals (Scientific Procedures) Act, 1986, and associated guidelines; EU Directive 2010/63/EU for animal experiments; or the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978). The study was approved by the Nanfang Hospital animal ethics committee (ID: NFYY-2016-107). In addition, every effort was made to minimize the number of animals used in the study and their sufferings.
2.6. Culture of pericytes After removing the neurointermediate lobe, anterior pituitary lobes from SD rats were dissociated into single cells using successive incubations in 0.25% Trypsin-EDTA. For pericytes, freshly dissociated cells were cultured in DMEM, containing 10% FBS at 37 °C and 5% CO2 waiting for pericytes crawling out. The pericytes were incubated with serum or serum-free medium for subsequent experiments. The medium was changed every 2–3 days. Positive identifcation of the cells was achieved by positive staining for desmin.
2.2. Antibodies The following antibodies were used in the study: desmin rabbit polyclonal antibody (ab32362; Abcam, Cambridge, UK), GH rabbit monoclonal antibody (ab134078, Abcam, Cambridge, England), α-SMA rabbit polyclonal antibody (ab5694, Abcam, Cambridge, England), 4,6diamidino-2-phenylindole (DAPI) (CAS 28718-90-3, Santa Cruz Biotechnology, Dallas, Texas, USA), type I collagen rabbit polyclonal antibody (abs131984, Absin, Shanghai, China), type III collagen rabbit polyclonal antibody (abs131560, Absin, Shanghai, China), Alexa Fluor 488 and 555-conjugated streptavidin secondary antibodies (Life Technologies, Carlsbad, California, USA), HRP goat anti-rabbit IgG (AS014, ABclonal, Wuhan, China), and GAPDH mouse monoclonal antibody (WHO79859, ABclonal, Wuhan, China).
2.7. Immunofluorescence The cells were seeded on Matrigel and poly-L-lysine-coated 12-mm glass coverslips in 24-well plates and allowed to attach for 5 days at 37 °C. They were then fixed in 4% paraformaldehyde (PFA) in PBS for 15 min at room temperature. They were permeabilized using 0.5% Triton X-100 (Solarbio, Beijing, China) in PBS for 10 min and blocked for 1 h with 5% BSA in PBS (both steps were done at room temperature). They were subsequently immunostained at 4 °C overnight with rabbit anti-rat desmin polyclonal antibody (1: 50 dilution) or rabbit anti-rat GH monoclonal antibody (1: 200 dilution) diluted in blocking buffer, followed by the incubation with secondary antibodies conjugated to Alexa Fluor 488 and 555-conjugated streptavidin secondary antibodies (1:500 dilution) for 1 h at room temperature. The coverslips were then washed with PBS and mounted on microscope slides with ProLong Gold Antifade Reagent containing DAPI to counterstain the nuclei. Samples were examined with a Leica DMI3000 B microscope.
2.3. Sampling SD rats were anesthetized with inhalation of isofluorane (5% induction; 2–3% maintenance) and euthanized by cervical dislocation. The adenohypophyses were dissected, mixed with phosphate-buffered saline (PBS), and then transferred to Biological Safety Cabinet rapidly. After dissecting, the adenohypophyses were randomly and equally divided into group A (improved method) and group B (traditional method).
2.8. Cell counting 2.4. Preparation of a single-cell suspension of somatotropic cells using the improved method
The images of somatotropic cells in group A or group B were taken using an inverted microscope (Leica DMI3000 B, Wetzlall, Germany) on days 1, 3, and 6. The somatotropic cells in images were analyzed using a TissueFAXS Plus flow-type tissue quantitative analyzer (TissueGnostics GmbH, Vienna, Austria).
Adenohypophyses were cut into pieces using a sterile tweezer and dispersed in 0.25% trypsin-EDTA (Gibco BRL, MD, USA) for 30 min at 37 °C. The cells were filtered through a 70-μm cell strainer first and then a 20-μm filter. Approximately 5.0 × 105 cells were usually obtained from each lobe. The cellular viability, as estimated using the trypan blue test, usually exceeded 90%. The cells were washed three times with PBS containing the antibiotics and antimycotics (Gibco BRL). Finally, the dispersed somatotropic cells were plated at a density of 3.0 × 105 onto 24-well culture plates in 1 mL of serum medium (DMEM/F12; Gibco BRL) supplemented with 10% FBS (Gemini BioProducts, California, USA) and 1% antibiotic–antimycotic solution.
2.9. Cell counting kit-8 analysis Cell counting kit-8 assay (CCK8; Dojindo, Kumamoto, Japan) was used to determine cell proliferation. Briefly, 60,000 cells per well were seeded into 96-well plates coated with poly-L-lysine. A group without cells served as the blank. Subsequently, 10 μL of CCK8 solution was added to cells in each well at different time points, followed by 94
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well of a Costar Transwell chamber (8 μM; Corning Life Sciences, MA, USA) in DMEM/F12. The latter were seeded in serum or serum-free medium, which was replaced with 10% FBS-containing medium after 24 h. The cells that had migrated to the bottom side of the membrane were fixed in 70% ethanol and stained with Giemsa stain solution 24 h after plating. Subsequently, the non-migrating cells in the upper chamber were removed using a cotton-tipped applicator. The membranes were mounted onto object slides, and six random fields per slide were counted with a 10× or 20× lens objective. At least three independent experiments were performed for each assay.
incubation at 37 °C for 1 h. The absorbance was measured at 450 nm on a microplate reader. The difference between the optical density (OD) of cells in the medium and that of blank (medium-only) wells is proportional with the survival/proliferation of cells. A curve was plotted for the change in cell activity with serum and serum-free media, using time as the x-axis and OD as the y-axis. 2.10. Enzyme-linked immunosorbent assay GH secretions of somatotropic cells cultured in group A and group B were compared using enzyme-linked immunosorbent assay (ELISA). Briefly, the cells were cultured in 24-well plates from day 1 to day 6. The cell supernatant was collected after each time point and kept at room temperature for 30 min, followed by centrifugation at 1000 rpm for 20 min. For fulfilling the criteria of ELISA kit (EZRMGH-45 K, Millipore, Burlington, Massachusetts, USA), the supernatant was concentrated using Pierce Protein Concentrator, 5 K (88534, Thermo Scientific, Waltham, Massachusetts, USA), following which GH in the supernatant was used immediately for ELISA analyses or, if not, aliquoted and stored at −80 °C for subsequent use. The samples were diluted and subjected to standard ELISA assays to determine GH levels in accordance with the manufacturer`s protocol.
2.14. Statistical analysis All experiments were run in triplicate. The results were expressed as mean ± standard deviation (SD). Data between groups were compared using the Student t test. Statistical analysis was conducted using SPSS 20.0 (IBM Corp., NY, USA). P values less than 0.05 were considered statistically significant. 3. Results 3.1. Comparison of the numbers of somatotropic cells in group A and group B
2.11. Western blot analysis Individual cells were released from the isolated adenohypophyses using trypsin/collagenase treatment. To compare the differences in the number of somatotropic cells between group A and group B with the extension of time, the microscopic images of cells were taken after 1 day, 3 days, and 6 days. As showed in Fig. 1A, spindle cells grew up only in group B on day 3. Then, immunofluorescence was performed to identify the two kinds of cells in group A and group B on day 3. GH was expressed only in group A, indicating that the cells in group A were pure somatotropic cells. But both desmin and GH were expressed in group B, indicating that the cells in group B were somatotropic cells mixed with pericytes (Fig. 1B). Therefore, the cell suspension was transferred in another plate for removing pericytes in group B. Next, the somatotropic cells in group A and group B were counted after 1 day, 3 days, and 6 days. The numbers of somatotropic cells in group A were almost constant after 6 days but decreased obviously in group B. On day 6, significantly more cells were found in group A than in group B (Fig. 1C; P < 0.01). Thus, compared with the traditional method, the improved method was more efficient for culturing primary somatotropic cells.
The cells were harvested, lysed in RIPA buffer (Thermo Scientific), and supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific). Protein extracts were subjected to centrifugation (12,000g) at 4 °C, and protein concentrations were determined using the BCA assay (Pierce). Protein was separated on either NativePAGE Bis-Tris Gel System or 4%–12% SDS-Tris-Glycine Protein Gel. Separated proteins were transferred onto polyvinylidene difluoride membranes (Millipore). The membranes were blocked with 5% nonfat dry milk in TBS and incubated with rabbit anti-rat α-SMA polyclonal antibody (1:100 dilution), rabbit anti-rat type III collagen polyclonal antibody (1:500 dilution) or rabbit anti-rat type I collagen polyclonal antibody (1:500 dilution). Subsequently, the membranes were washed and incubated with appropriate secondary, HRP goat anti-rabbit IgG (1:1000 dilution). Proteins were visualized using autoradiography and enhanced chemiluminescence (ECL; Amersham Biosciences, GE Healthcare). 2.12. Scratch wound healing assay
3.2. Comparison of the status of pericytes in serum-free and serum media
The migration of pericytes in serum or serum-free medium was assessed using the scratch wound healing assay proposed by Rodriguez et al (Rodriguez et al., 2005). Briefly, the cells (3 × 105) were seeded into a six-well plate in 2 mL of serum medium and antibiotics. The cells were incubated at 37 °C and in a 5% CO2 atmosphere until confluence. Once confluent, monolayer cells in each dish were subjected to a mechanical wound by scratching with a 200-μL pipette tip, parallel to the grid bars along the central grid line. Wound placement was checked with a Leica microscope (Leica DMI3000 B). The medium was removed and the cells were washed five times with PBS, followed by the serum or serum-free treatments. The cells along the induced wound in each dish were taken after 0 and 24 h using a digital camera (Olympus Camedia 34–40 zoom, 5× magnification) attached to a light microscope. Computer program Image J (National Institutes of Health, MD, USA) was used to measure the area of initial damage (images taken at time 0) and the remaining damage after 24 h. The percentage of wound repair was calculated.
Next, the study investigated the established effect of the serum and serum-free medium on pericytes because pericytes grew up only in group B. Firstly, these cells obtained from adenohypophysis specimens were identified as pericytes by positive staining for desmin (Fig. 2A) (Tsukada et al., 2016). The results of Western blot analysis indicated that compared with the serum medium, the expression of ɑ-SMA (a marker of activating pericytes), type I collagen, and type III collagen was poor in the serum-free medium (Fig. 2B). The proliferation of pericytes was examined using CCK8 analysis. The OD of pericytes was significantly lower in the serum-free medium than in the serum medium after 24 h which indicated a lower proliferative activity of pericytes in the serum-free medium (Fig. 2C; P < 0.001). The study then investigated whether the modulation of the serum-free medium affected the migration of pericytes. None of the pericytes passed through the chamber in the serum-free medium in Boyden chamber assays (Fig. 2D). Similarly, as shown in Fig. 2E and F, the ratio of scratch wound healing was lower when pericytes were cultured in the serum-free medium compared with the serum medium (P < 0.001). These findings indicated that compared with the serum medium, the serum-free medium did not support the survival of pericytes.
2.13. Migration assay The cell migration was assessed using a Boyden chamber assay. For these experiments, 1 × 105 cells (pericytes) were seeded onto the upper 95
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Fig. 1. Differences in the numbers of somatotropic cells between group A and group B at different time points. A. Microscopic images of somatotropic cells were taken on days 1, 3, and 6. Spindle cells grew up in group B on the third day. The medium was changed for wiping off spindle cells in group B. Red arrow: somatotropic cells. Green arrow: spindle cells. Scale bar = 100 μm. B. Cells in group A and group B on the third day were identified by immunofluorescence. The cells in group A only expressed GH (red), indicating that the cells in group A were pure somatotropic cells. The cells in group B expressed GH (red) and desmin (green), indicating that the cells in group B were somatotropic cells mixed with pericytes. DAPI (blue stain) was used as a nuclear marker. Scale bar = 20 μm. C. A representative graph for cell counts of somatotropic cells in group A and group B (n = 3/group) on days 1, 3, and 6. Data are shown as mean ± SD. *P < 0.05; **P < 0.01.
Fig. 2. Comparison of the growth status of pericytes in the serum and serum-free media for 24 h. A. Identification of primary pericytes in culture. Pericytes are characterized by positive staining for desmin (green). DAPI (blue stain) was used as a nuclear marker. Scale bar = 20 μm. B. Western blot analysis was used to detect the expression of ɑ-SMA, type I collagen, and type III collagen in pericytes. GAPDH expression was monitored to confirm equal protein loading and blotting. C. Detecting the viability of pericytes cultured in the serum and serum-free media using CCK8 assay (n = 3/group). Data are shown as mean ± SD. **P < 0.01; ***P < 0.001. D. Cell migration analysis of pericytes cultured in serum and serum-free media as evaluated using Boyden chamber assay. Scale bar = 100 μm. E. Scratch wound healing assay of pericytes after indicated treatments for 24 h. Scale bar = 100 μm. F. Statistical analyses of the scratch wound area of pericytes are shown (n = 3/group). Data are shown as mean ± SD. ***P < 0.001. 96
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Fig. 3. Comparison of the status of somatotropic cells in group A and group B within 6 days. A. CCK8 assay was used to detect the viability of somatotropic cells in group A and group B (n = 3/group). Data are shown as mean ± SD. **P < 0.01; ***P < 0.001. B. ELISA assay was used to analyze the GH secretion in somatotropic cell supernatant in group A and group B (n = 3/group). Data are shown as mean ± SD. **P < 0.01; ***P < 0.001.
treatments had much higher cell isolation rates than collagenase treatment, with no damage to somatotropic cells. Double filtration was used for removing pericytes, which decreased the number of pericytes at the beginning of the culture. Moreover, the components in the culture medium (DMEM/F12 containing 0.5% BSA, B27, and N2 supplements besides 20 ng/mL bFGF and EGF) were optimized and the presence of somatotropic cells was confirmed. The main obstacle to establishing pure somatotropic cells in vitro is pericytes contamination of the cultures. In the present study, somatotropic cells in the adenohypophysis were isolated by two-step filtration and cultured in the serum-free medium. The traditional method usually involves filtering the harvested tissues using a 70-μm cell strainer for obtaining the cell suspension. However, it cannot separate the pericytes from somatotropic cells. The diameters of pericytes and somatotropic cells are different. After many experiments, it was found that somatotropic cells could pass through a 20-μm cell strainer, whereas pericytes could not. The number of pericytes was minimized when a single-cell suspension was obtained by two-step filtration. FBS is the supplement most widely used for cell culture. It provides hormones, proteins, minerals, and several other factors (Paula et al., 2015). The pericytes show superior growth and gradually replace somatotropic cells in the serum medium. The mode of separation used in the traditional method is not preferred because it is associated with the loss of somatotropic cells. The main reason is that FBS strongly supports the growth of pericytes, as reported earlier (Shahdadfar et al., 2005). Therefore, finding the substitutes of FBS is essential to ensure cell manipulation. Developing a chemically defined cell culture condition that supports the growth of somatotropic cells but not of pericytes is an important issue. Different studies have attempted to replace FBS with human-derived alternatives (Aldahmash et al., 2011; Bernardo et al., 2007; Bieback et al., 2009; Kocaoemer et al., 2007; Lindroos et al., 2010; Shahdadfar et al., 2005) or serum-free defined media (Agata et al., 2009; Chase et al., 2012; Mimura et al., 2011; Rodrigues et al., 2010; Tarle et al., 2011; van der Valk et al., 2010). Therefore, finding a medium formula suitable for somatotropic cells but not for pericytes is possible. The serum-free defined media comprise synthetic supplements that replace serum and reduce variability, thus offering the most promising alternative. Thus, this study proposed the use of N2/B27/bFGF/EGF, which was the regular supplement for culturing the primary cells in the serum-free medium (Chen et al., 2016; Fauquier et al., 2008). The culture of primary somatotropic cells using the medium with N2/B27/bFGF/EGF was successful, and the morphologic characteristics of somatotropic cells cultured in serum-free medium were comparable to those of cells in FBS. The somatotropic cells in group A were rounded, bright, and floating; they formed several colonies. However, the somatotropic cells in group B had a pericytes-like shape, reduced brightness, and tendency to cluster at the confluence. These observations agreed with another published study that revealed similar
3.3. Comparison of the status of somatotropic cells in group A and group B To assess the status of somatotropic cells in group A and group B, CCK8 assay was performed to detect the viability of somatotropic cells in the two groups. 60,000 cells were plated per well from the two groups. The result indicated that the viability of somatotropic cells in group A was quite stable within 6 days, but it decreased in group B (Fig. 3A; P < 0.001). Next, ELISA assay was performed to detect GH secretion of somatotropic cells in group A and group B. The same number cells were cultured in plates from day 1 to day 6. And then the cell supernatant was collected. The result showed that the GH secretion of somatotropic cells in group B increased within 3 days and then remained constant, indicating that somatotropic cells in group B could not secrete GH after 3 days. On the contrary, the GH secretion of somatotropic cells in group A increased within 6 days, indicating that somatotropic cells in group A secreted GH continuously. After 6 days, the GH secretion of somatotropic cells was significantly higher in group A than in group B (Fig. 3B; P < 0.001). These results collectively indicated that somatotropic cells in group A were in a better status compared with group B. 4. Discussion The mechanisms underlying adenohypophysis and neuroendocrine regulation have gained immense attention in recent years. Many immortalized cell lines that secrete various kinds of hormones have been developed to meet the research needs (Ooi et al., 2004). However, the transformed immortalized cell lines generally grow well under in vitro culture conditions, thus exhibiting limited differentiation properties compared with their wild-type counterparts. Also, the pathophysiological mechanism may differ from the mechanism for primary cultured cells. Therefore, the use of primary cultured cells is important for studying their differentiation properties (Ooi et al., 2004). Nevertheless, somatotropic cells are not capable of propagating and pericytes contamination in primary culture is a common problem (Kassen et al., 1996; Vidrich et al., 1988). Therefore, culturing primary somatotropic cells without pericytes contamination needs further exploring. This study aimed to propose a method for culturing somatotropic cells. The use of the improved and traditional methods for isolating and culturing somatotropic cells in vitro was evaluated. In the present study, an improved method was developed to isolate somatotropic cells, and the culturing conditions required for the survival of somatotropic cells were determined. The adenohypophysis was chosen rather than the entire pituitary gland because somatotropic cells are located in the adenohypophysis (Gajkowska et al., 2006), thereby providing sufficient pure cells. In addition, both enzyme digestion and tissue culture methods were used to isolate primary somatotropic cells, hence greatly improving the rate of obtaining primary somatotropic cells. The previous method was modified by cutting the adenohypophysis into pieces and digesting it with 0.25% trypsin-EDTA because trypsin-EDTA 97
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characteristics (Kokubu et al., 2015). Further, differences in the survival rates of somatotropic cells were observed under different culture conditions. The results indicated that the culture conditions had a marked effect after 3 days; the serum-free medium provided a better survival rate for somatotropic cells. Conversely, the viability of pericytes in the serum-free medium used in group A, compared with the serum medium, was significantly inhibited. Therefore, these methods could solve the problem of pericytes contamination successfully.
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5. Conclusion This study proposed a more rational method for obtaining single, intact primary somatotropic cells that were of good quality in terms of survival rate, integrity, and general morphology. The improved method helped avoid pericytes contamination. The primary somatotropic cells were not readily stimulated and the risk of contamination was minimized because the cells needed no purification again in subsequent cultures. The findings might provide a solid foundation for studying the biological characteristics of GH. This method can be refer to other studies on primary cultures contaminated with pericytes. Funding This study was supported by National Science and Technology Infrastructure Program (2014BAI04B01), Scienceand Technology Planning Project of Guangdong Province, China (2016A020213006) (2017A030303021), Chinese National Natural Science Foundation (81701197), Natural Science Foundation of Guangdong Province (2017A030310111), President Foundation of Nanfang Hospital, Southern Medical University (2017B009), Clinical Research Startup Program of Southern Medical University by High-level University Construction Funding of Guangdong Provincial Department of Education (LC2016PY012). Authors contributions Jian Mao, Yun Bao, Fen Mei performed the experiments. Xixian Liao, Fan Liu wrote the paper. Lizhi Zhou performed data analysis. Songtao Qi, Binghui Qiu conceived the ideas and designed the experiments Conflict of interest The authors declare that they have no conflict of interests. References Agata, H., Watanabe, N., Ishii, Y., Kubo, N., Ohshima, S., Yamazaki, M., Tojo, A., Kagami, H., 2009. Feasibility and efficacy of bone tissue engineering using human bone marrow stromal cells cultivated in serum-free conditions. Biochem. Biophys. Res. Commun. 382 (2), 353–358. Aldahmash, A., Haack-Sorensen, M., Al-Nbaheen, M., Harkness, L., Abdallah, B.M., Kassem, M., 2011. Human serum is as efficient as fetal bovine serum in supporting proliferation and differentiation of human multipotent stromal (mesenchymal) stem cells in vitro and in vivo. Stem Cell Rev. 7 (4), 860–868. Balen, A.H., Er, J., Rafferty, B., Rose, M., 1995. Characterization of a rat anterior pituitary cell bioassay. In Vitro Cell. Dev. Biol. Anim. 31 (4), 316–322. Bartke, A., Darcy, J., 2017. GH and ageing: pitfalls and new insights. Best Pract. Res. Clin. Endocrinol. Metab. 31 (1), 113–125. Bernardo, M.E., Avanzini, M.A., Perotti, C., Cometa, A.M., Moretta, A., Lenta, E., Del Fante, C., Novara, F., de Silvestri, A., Amendola, G., Zuffardi, O., Maccario, R., Locatelli, F., 2007. Optimization of in vitro expansion of human multipotent mesenchymal stromal cells for cell-therapy approaches: further insights in the search for a fetal calf serum substitute. J. Cell. Physiol. 211 (1), 121–130. Bieback, K., Hecker, A., Kocaomer, A., Lannert, H., Schallmoser, K., Strunk, D., Kluter, H., 2009. Human alternatives to fetal bovine serum for the expansion of mesenchymal stromal cells from bone marrow. Stem Cells 27 (9), 2331–2341. Bijkerk, R., Au, Y.W., Stam, W., Duijs, J., Koudijs, A., Lievers, E., Rabelink, T.J., van Zonneveld, A.J., 2019. Long non-coding RNAs Rian and miat mediate myofibroblast formation in kidney fibrosis. Front. Pharmacol. 10, 215. Chase, L.G., Yang, S., Zachar, V., Yang, Z., Lakshmipathy, U., Bradford, J., Boucher, S.E.,
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