Data on isolating mesenchymal stromal cells from human adipose tissue using a collagenase-free method

The present dataset describes a detailed protocol to isolate mesenchymal cells from human fat without the use of collagenase. Human fat specimen, surgically cleaned from non-fat tissues (e.g., blood vessels) and reduced into smaller fat pieces of around 1–3 mm size, is incubated in complete culture media for five to seven days. Then, cells started to spread out from the fat explants and to grow in cultures according to an exponential pattern. Our data showed that primary mesenchymal cells presenting heterogeneous morphology start to acquire more homogenous fibroblastic-like shape when cultured for longer duration or when subcultured into new flasks. Cell isolation efficiency as well as cell doubling time were also calculated throughout the culturing experimentations and illustrated in a separate figure thereafter. This paper contains data previously considered as an alternative protocol to isolate adipose-derived mesenchymal stem cell published in “Proliferation and differentiation of human adipose-derived mesenchymal stem cells (ASCs) into osteoblastic lineage are passage dependent” [1].


a b s t r a c t
The present dataset describes a detailed protocol to isolate mesenchymal cells from human fat without the use of collagenase. Human fat specimen, surgically cleaned from non-fat tissues (e.g., blood vessels) and reduced into smaller fat pieces of around 1-3 mm size, is incubated in complete culture media for five to seven days. Then, cells started to spread out from the fat explants and to grow in cultures according to an exponential pattern. Our data showed that primary mesenchymal cells presenting heterogeneous morphology start to acquire more homogenous fibroblastic-like shape when cultured for longer duration or when subcultured into new flasks. Cell isolation efficiency as well as cell doubling time were also calculated throughout the culturing experimentations and illustrated in a separate figure thereafter. This paper contains data previously considered as an alternative protocol to isolate adipose-derived mesenchymal stem cell published in "Proliferation and differentiation of human adipose-derived mesenchymal stem cells (ASCs) into osteoblastic lineage are passage dependent" [1]. &

Subject area
Cell Biology More specific subject area Cell culture, mesenchymal stromal cells Type of data Image (microscopy), text file, graph, figure How data was acquired Cell counting using light microscope, statistical analysis, math formula calculation Data format Raw and analyzed data Experimental factors Cells isolated from fat explants cultured in plastic flasks

Experimental features
Spontaneous isolation of mesenchymal cell using in vitro cell culture system and without the use of collagenase Data source location Lebanese American University, Byblos, Lebanon Data accessibility Data are provided in the article

Value of the data
The below data provide a detailed and reproducible collagenase-free protocol to isolate mesenchymal stromal cells from human adipose tissue.
These data enable researchers to isolate various cell types populating fat. These data offer the possibility to isolate specific primary cell cultures with a reduced and efficient cell isolation yield.

Data
The data presented in this paper correspond to the isolation of mesenchymal stromal cells without digesting human fat pieces with collagenase. Subsequently, cell morphology, primary cell isolation efficiency as well as cell population doubling time were measured using light microscopy analysis and

. Fat specimen
Fresh fat specimen obtained from surgery is incubated in a sterile saline solution at room temperature and processed within 2-4 h postsurgery. Procedures were approved by the Institutional Review Board (IRB) of the Lebanese American University. Patients were chosen not to have chronic diseases or cancers. Abdominal adipose tissue resected from middle aged men patients aged 40-50 years old and were asked to read the consent forms and approve/sign them prior to surgery. Primary cells isolated from different donors were mixed together and then subcultured according to the protocol described below.

Protocol of primary cell isolation and culture
Five grams of human fat are incubated in 40 ml of warm sterile PBS (1 Â ) containing 3% Pen/Strep for 30 min. Then extra non-fat tissue (e.g., blood vessels and connective tissues...) were surgically removed under sterile conditions. One gram of tissue is minced into pieces of 8-10 mm 3 under sterile conditions. Minced fat pieces were equally distributed into two T70cm 2 cell culture plates (0.5 g of fat/ plate) containing each 5 ml of complete growth medium [RPMI þ10% FBS þ1% penicillin/streptomycin (100 U/ml penicillin, 100 mg/ml streptomycin)] and incubated in CO 2 cell culture incubator at 37°C and left undisturbed for 3 days. After 3 days of culture the growth media is replaced with a new fresh growth media every 3 days by gently bending the plate and aspirating the old media with a sterile pipet. On day six the explants cultures can be observed under light microscopy and cells appear to spread out of the fat pieces into the surroundings of the fat specimens. At day 9, fat pieces are removed with a sterile pipet and the cell cultures were then washed twice with 5 ml of sterile warm PBS (1 Â ) and finally cultured with 10 ml of new fresh complete growth medium until near confluency for further experimental analysis.

Cell splitting
Cell splitting is done using a near confluent plate. First the plate is washed twice with 5 ml of sterile warm PBS (1 Â ), then 3 ml of warm sterile trypsin (1 Â ) are added, after discarding the wash buffer, and the plate is incubated in a CO 2 cell culture incubator at 37°C for 2-3 min. After adding 10 ml of complete growth media to inhibit trypsin effect, the medium is collected and transferred into sterile 50 ml conical tube, the remaining cells in the plate can be collected by an additional washing with 2 ml of sterile warm PBS (1 Â ). The cells are separated by centrifugation at 2000 RPM for 5 min at room temperature. The supernatant is discarded while the pellet is resuspended with the desired volume complete fresh growth medium. Finally, cells are counted using a hemocytometer and appropriate number of cells are seeded as desired for the experimentation.

Primary cell isolation efficiency (CIE) calculation
To measure the primary cell isolation efficiency (CIE) of cells isolated from fat explants, primary cells remaining after removing fat and subsequent wash are trypsinized, manually counted on a hemocytometer with trypan blue exclusion method and cell stained with trypan were excluded from the count. Then, CIE is calculated according to the following mathematical formula: CIE ¼ [(number of primary cells obtained immediately after removing fat pieces)/(number of primary days in culture)]/ (total mass of fat explant in grams). Number of primary cells obtained immediately after removing fat pieces ¼ 3 Â 10 5 ; number of primary culture days ¼ 9; total mass of fat explant¼ 0.5 g. CIE¼ 66,000/ gram tissue.

Growth kinetic protocol and calculation of cell population doubling time
To measure growth kinetic of cell cultures isolated from explants, primary mesenchymal cells remained after removing fat pieces and subsequent wash, are trypsinized, manually counted on a hemocytometer with trypan blue exclusion method and cells stained with trypan were excluded from the count. Trypsinized primary cells were plated at a density of 3 Â 10 3 cells per cm 2 in 6-well plates to make a total of 30 wells. Cells were then kept overnight to adhere and maintained for ten days with medium changes occurring every two days starting the day after plating (day 0) and continuing each day until day 9. Three dishes were trypsinized every day to determine cell number and viability using trypan blue exclusion methods and cells stained blue with trypan were excluded from the count. Cell population doubling time (DT) of cultured mesenchymal cells was calculated using the following formula: DT¼(t Â Ln 2)/(Ln N2 À Ln N1), t ¼number of days in culture, N1 ¼number of cells at the beginning of the log phase, N2 ¼number of cells at the end of the log phase. N1¼35.500 72784 correspond to the number of cells at day 2, and N2 ¼83,000 71528 correspond to the number of cells at day 7, then DT¼ 4.12 70.4.

Statistics
All numerical data are presented as mean 7standard error. Statistics analysis is done using t test. P value of less than 0.05 was considered to be statistically significant. Differences were evaluated by one-way analysis of variance (ANOVA). Differences were considered significant at (*P o0.05). All experiments were done in triplicate and data were expressed as mean 7SEM.