{"id":359792,"date":"2023-11-24T19:00:00","date_gmt":"2023-11-25T00:00:00","guid":{"rendered":"https:\/\/platohealth.ai\/an-immortal-porcine-preadipocyte-cell-strain-for-efficient-production-of-cell-cultured-fat-communications-biology\/"},"modified":"2023-11-25T15:30:21","modified_gmt":"2023-11-25T20:30:21","slug":"an-immortal-porcine-preadipocyte-cell-strain-for-efficient-production-of-cell-cultured-fat-communications-biology","status":"publish","type":"post","link":"https:\/\/platohealth.ai\/an-immortal-porcine-preadipocyte-cell-strain-for-efficient-production-of-cell-cultured-fat-communications-biology\/","title":{"rendered":"An immortal porcine preadipocyte cell strain for efficient production of cell-cultured fat – Communications Biology","gt_translate_keys":[{"key":"rendered","format":"text"}]},"content":{"rendered":"
<\/div>\n

Cell culture<\/h3>\n

ISP-4, C2C12, and immortalized porcine muscle satellite cell (PMSC, a kindly gift from Professor Li-Min Hou, Nanjing Agriculture University) were cultured in growth medium (GM), containing Dulbecco\u2019s modified Eagle\u2019s medium (Servicebio#G4511), 10% fetal bovine serum (Procell#164210-50), 100U\/ml penicillin (Sangon#A610028), 100\u2009\u00b5g\/ml streptomycin (Sangon#A610494). For PMSC, an extra 10% FBS and 2\u2009ng\/mL FGFb (Novoprotein#C046) were added.<\/p>\n

ISP-4 adipogenic differentiation medium (ADM) consisted of growth medium supplemented with 1\u2009\u00b5g\/mL Insulin (Novolin R), 5\u2009nM 3,3\u2019,5-Triiodo-l<\/span>-thyronine (Yuanye Bio-Technology#S24025-25mg), 2\u2009\u00b5g\/ml dexamethasone (Sangon#A601187), 100\u2009\u00b5M 3-isobutyl-1-methylxanthine (IBMX, Sangon#A606630), 125\u2009\u00b5M indomethacin (Makclin#I811784), 1\u2009\u00b5M rosiglitazone (Makclin#R832516), 33\u2009\u00b5M biotin (Makclin#B6220), 17\u2009\u00b5M Pantothenic acid (Sangon#A600683), 1\u2009\u00b5g\/\u00b5l Transferrin (Yuanye Bio-Technology #S12027). ISP-4 adipogenic maintenance medium (AMM) includes growth medium supplemented with 1\u2009\u00b5g\/mL Insulin (Novolin R).<\/p>\n

For the 4\u2009+\u20094 protocol, the cells were first recovered in a growth medium for 2 days, followed by a switch to ADM for 4 days. Afterward, the cells were maintained in AMM for an additional four days, mediums were changed every 2 days.<\/p>\n

For the 2\u2009\u00d7\u20095 protocol, the cells were seeded and cultured in AMM for 10 days, with medium changes every 2 days.<\/p>\n

Cell culture in alginate hydrogel<\/h3>\n

To prepare the cell\/alginate suspension, ISP-4 cells were resuspended in a 0.75% alginate solution (Sigma#W201502) at a concentration of 1.25\u2009\u00d7\u2009107<\/sup> cells\/ml. The suspension was then injected into a solidification buffer (50\u2009mM CaCl2<\/sub>) and placed in a 37\u2009\u00b0C incubator for 20\u2009minutes to solidify into microfibers. After washed with DMEM, the microfibers were transferred into a growth medium (GM) for recovery before adipogenesis according to different protocols.<\/p>\n

Cell culture in wire-drawing protein scaffold<\/h3>\n

Irradiated sterilized wire-drawing peanut protein was soaked in growth medium overnight and then cut into small cubes (about 1\u2009cm2<\/sup> in area and 0.3\u2009cm in thickness). These cubes were subsequently dried using sterile filter paper and placed onto a 10\u2009cm dish for later use.<\/p>\n

To seed cells onto the scaffolds, 2.5\u2009\u00d7\u2009106<\/sup> ISP-4 were resuspended in 7\u2009\u00b5l thrombin (20NIH units per ml, Biosharp#BS903). Next, 7\u2009\u00b5l fibrinogen (15\u2009mg\/ml, Biosharp#BS943) was added immediately before seeding the cells onto the scaffolds. The cell blend was mixed well and dropped onto the scaffolds. The dish with seeded scaffolds was then placed into a 37\u2009\u00b0C incubator for 20\u2009minutes to allow the fibrin to gel. Once gelation had occurred, a growth medium was added to the dish, and the scaffold with cells was cultured as indicated.<\/p>\n

RNA extraction, cDNA synthesis, and RT-qPCR<\/h3>\n

RNA isolation was performed using Total RNA Extraction Reagent (Vazyme#R401), followed by reverse transcription using HiScript II Q RT SuperMix (Vazyme#R223), as per the manufacturer\u2019s guidelines. Real-time quantitative PCR (RT-qPCR) was carried out with iQ SYBR Green Supermix (Servicebio#G3326) using the QuantStudio\u2122 5 System (ThermoFisherScientific). The sequence of primers is listed in supplementary Table\u00a01<\/a>. The 2\u2212\u2206\u2206ct<\/sup> method was used to calculate relative gene expression, with 36B4 as the reference gene for normalization.<\/p>\n

Fluorescence staining<\/h3>\n

For 2D cultured ISP-4 or mixed culture cells of ISP-4 and C2C12\/PMSC, samples were fixed in 4% paraformaldehyde. After washing with PBS, fixed cells were permeabilized with 0.2% Triton X-100, then blocked with 3% BSA solution, anti-DESMIN (Abclonal, Cat#A0699, Lot#5500004718) and anti-MYL1 (Servicebio, Cat#GB112474, Lot#AC230928017) were used to detect mature muscle cells. After being washed with PBS, cells were re-stained within Goat Anti-Rabbit IgG-DyLight 549 (Bioworld, Cat#BS10023, Lot#CL89330), Hoechst33342 (1:1000, Beyotime#C1028), and BODIPY FL (1:5000, Thermo Fisher#D3922).<\/p>\n

For alginate hydrogel, samples were fixed in 4% paraformaldehyde containing 50\u2009mM CaCl2<\/sub>. The samples were stained with Hoechst33342 (1:1000, Beyotime#C1028), BODIPY FL (1:5000, Thermo Fisher#D3922) and Actin-Tracker Red-Rhodamine (1:200, Beyotime#C2207S) in PBS for 30\u2009min. And the samples were washed with PBS before imaging.<\/p>\n

ISP-4 and C2C12\/PMSC mixed culture<\/h3>\n

For coculture with C2C12, a suspension of ISP-4 cells (1\u2009\u00d7\u2009105<\/sup> cells) and C2C12 cells (3\u2009\u00d7\u2009105<\/sup> cells) was seeded in six-well plates containing growth medium. The cells were then cultured for 48\u2009h. Subsequently, differentiation was initiated by switching to ADM, and after 4 days, the medium was exchanged into AMM for another 4 days. Then the medium was changed into AMM with 2% horse serum to initiate myogenesis.<\/p>\n

For coculture with PMSC, 0.6\u2009\u00d7\u2009105<\/sup> ISP-4 cells and 1.4\u2009\u00d7\u2009105<\/sup> PMSC cells were seeded into a 12-well plate with 1\u2009mL culture medium. After 24\u2009h recovery, cells were differentiated with ADM for 48\u2009h, and AMM for another 48\u2009h. Then cells were cultured in AMM with 2% horse serum for the other 4 days.<\/p>\n

The cells were maintained in a 5% CO2<\/sub> humidified incubator at 37\u2009\u00b0C, with medium exchanges every 2 days.<\/p>\n

Microcarrier cell culture<\/h3>\n

100\u2009mg of 3D TableTrix microcarriers (CytoNiche) were dissolved in 20\u2009mL of growth medium in a 125\u2009mL sterile spinner flask (CytoNiche) to obtain a final concentration of 5\u2009mg\/mL. The mixture was left to dissolve overnight at 4\u2009\u00b0C. The next day, the spinner flasks were prewarmed to 37\u2009\u00b0C, and a total of 2.5\u2009\u00d7\u2009106<\/sup> ISP-4 cells in 30\u2009ml growth medium were added.<\/p>\n

The spinner flasks were positioned on magnetic stirring platforms in the incubator. For the first 24\u2009h, rotation speed was set to 35\u2009rpm for 5\u2009min, then followed by 0\u2009rpm for 1\u2009h, and repeated 24 times. Afterward, the rotation mode was changed to a constant speed of 40\u2009rpm. 50% growth medium was changed every 48\u2009h.<\/p>\n

For cell counting, 1\u2009mL of medium containing microcarriers was collected from a spinner flask. 200\u2009\u00b5L of supernatant was removed and replaced with lysate (CytoNiche). After incubating at 37\u2009\u00b0C for 30\u2009min, the cells were then counted using a hemocytometer,<\/p>\n

To assess the differentiated microcarriers in culture flasks, 1\u2009mL of cell suspension was pipetted and stained with Hoechst33342 (1:1000, Beyotime, C1028) and BODIPY FL (1:5000, Thermo Fisher, D3922).<\/p>\n

Area of lipid droplet analysis<\/h3>\n

All images were taken by confocal laser scanning microscope (LSM750, Zeiss) under the same acquisition conditions. Images were processed in ImageJ (version: 2.1.0\/1.53c) as the following step. 1. Scale bar calibration: Open the image with only BODIPY staining, measure the length of the image\u2019s scale bar, and set the pixel-to-length (\u00b5m) ratio in \u201cset scale\u201d. 2. Fluorescence area measurement: Convert the image to \u201c8-bit\u201d, open \u201cThreshold\u201d, adjust the threshold, select the green fluorescent area, and select \u201cMeasure\u201d to obtain the area of the lipid droplets. 3. Cell nuclei counting: open the image with only nucleus staining, and use ImageJ\u2019s \u201canalyze particles\u201d tool to count the cell nuclei stained with Hoechst.<\/p>\n

Oil-red staining<\/h3>\n

Cells were fixed with 4% PFA at 4\u2009\u00b0C overnight. Following fixation, the cells were rinsed with 60% isopropanol and then stained with Oil Red O (0.42\u2009g\/mL in 60% isopropanol) for 5\u2009min. The Oil Red O solution was subsequently removed, and the cells were washed three times with tap water. The Oil Red O stain was then extracted using isopropanol and the absorbance was measured at 500\u2009nm.<\/p>\n

Western blot<\/h3>\n

Protein was extracted with RIPA (Servicebio#G2002), target proteins were detected with antibodies as follow: anti-PERILIPIN 1 (Bioss, Cat#bs-3789R, Lot#AD121123), anti-DESMIN (Abclonal, Cat#A0699, Lot#5500004718), Anti-FLAG (Genscript, Cat#A00187, Lot#155000952), Anti-GAPDH (Bioworld, Cat#AP0063, Lot#AA55151), anti-TUBULIN (Sangon, Cat#D190090-0100, Lot#H508AA0104).<\/p>\n

Lipid content analysis<\/h3>\n

The lipid content was measured using a triglyceride content assay kit (Sangon#D799795), based on colorimetric method, following the manufacturer\u2019s instructions. For sample preparation, ~0.1\u2009g of hydrogel or microcarrier was homogenized using a blue pestle in 1\u2009ml Buffer 1. Then, 200\u2009\u00b5L of supernatant was collected for further analysis. 200\u2009\u00b5L solution with 1\u2009mg\/mL triglyceride was used for comparison. The lipid content was calculated using the following formula.<\/p>\n

\n

$${rm {L{ipid}}},{rm {{content}}}=\tleft(1,{rm {{mg}}}\/{rm {{mL}}}times 0.2,{rm {{mL}}}right) \t times frac{{A}^{{rm {{sample}}}}-{A}^{{rm {{Blank}}}}}{{A}^{{{rm {stander}}}}-{A}^{{{rm {Blank}}}}}\/,{{rm {sample}}; {rm {weight}}},({rm {g}})$$<\/span><\/p>\n<\/div>\n

Statistics and reproducibility<\/h3>\n

Statistical analysis was performed using Prism 9.0 (GraphPad). Analysis between two groups was performed using a Two-tailed unpaired Student\u2019s t<\/i>-test. Sample size has been indicated in each figure legend.<\/p>\n

Reporting summary<\/h3>\n

Further information on research design is available in the\u00a0Nature Portfolio Reporting Summary<\/a> linked to this article.<\/p>\n