5.7.1 Effect of body fluids on adipogenesis
10 % of LAT fluid together with 10 % paired serum samples in fetal bovine serum (FBS) - free PM4 medium were applied directly on ‘standard’ preadipocyte culture with 70 % confluence analyse adipogenic effect of preadipocytes. The medium supplemented with body fluids was changed twice. Preadipocytes were then subjected to standard adipogenic procedure for 3 days. Both preadipocytes and 3-days differentiated adipocytes were lysed for gene expression analysis.
5.7.2 Single and coculture models
To study in vitro direct and indirect interconnections between adipocytes and HDLECs, we have developed four single and coculture models (Fig. 12).
60 Figure 12: 2D in vitro single and coculture models of HDLECs and white adipocytes are depicted schematically. (A) Single culture model: generating adipose conditioned medium (ACM) from lipolytic adipocytes was applied directly to HDLECs. (B) Single-culture model: lymphatic CM (LCM) derived from HDLECs was directly applied to white adipocytes. (C) An indirect system using an insert system was designed between HDLECs and white adipocytes. (D) A direct system was designed between HDLECs and white adipocytes.
5.7.2.1 Single culture: Application of ACM on HDLECs
Collection of adipose conditioned medium (CM) - 12 day differentiated adipocytes were washed twice with DPBS +Mg2+ and Ca2+ and insulin starved in EBM-2 medium supplemented with transferrin (0.1 ug/ml) for 16–19 h. The medium was aspirated, and cells were exposed to EBM2 (supplemented with transferrin (0.1 ug/ml), 0.5% free FFA, and endotoxin low BSA) with or without 1 mM dibutyryl-cAMP (lipolysis inducer) for 30 min.
Then the medium was discarded, and cells were washed twice with DPBS +Mg2+ and Ca2+
to remove any traces of dibutyryl-cAMP. Then fresh EBM2 (supplemented with transferrin (0.1 ug/ml), 0.5 % FFA free and endotoxin low BSA) was added for 4 h incubation. After 4 h, the ACM (i.e., CM-L from cells stimulated with dibutyryl-cAMP and CM-B from unstimulated cells) was collected under aseptic conditions, centrifuged to remove any released cells, and then filtered using a filter unit NalgeneTM syringe with a 0.2 µm membrane to remove cell debris. EBM2 medium without inducer, which was handled in the same way as conditioned medium but was not in contact with cells, was used as a control (CON). Media were stored at -80 °C until use.
61 The ACM was applied to HDLECs-cells at a density of 1.9 x 104 cells/cm2 using the standard subcultivation protocol. Once the cells reached 50 % confluence, they were washed twice with DPBS +Mg2+ and Ca2+ and 0.5 ml of ACM was added for 24 and 48 hours. After 1 day, 2 days cells were washed once with PBS and then lysed with RLT lysis buffer (200 ul/per well) and stored at -80 ° C for gene expression analysis.
5.7.2.2 Single culture: Application of LCM on white adipocytes
A collection of LCM - HDLECs were grown in EGM-2 medium supplemented with 5 % fetal bovine serum until 100 % confluence. LCM was collected after 2 days and under aseptic conditions, centrifuged to remove any released cells and then filtered using NalgeneTM syringe with a 0.2 µm membrane to remove cell debris. Media were stored at – 80°C until use.
LCM was applied to white, fully differentiated adipocytes (standard culture, male donors) that had been washed twice with DPBS+Mg2+ and Ca2+ and insulin starved in DMEM/HAM's F-12 supplemented with transferrin (0.1 ug/ml) for 16-19 hours. Then medium was aspirated, and cells were exposed to LCM (diluted with EBM-2 medium in a ratio of 1:1) for 42 hours. EGM-2 medium diluted with EBM-2 medium was used as control.
After exposition to LCM and control media, cells were washed once with DPBS +Mg2+ and Ca2+and used for a standard lipolytic assay described in 5.8.
5.7.2.3 Indirect coculture system
Fully differentiated ‘standard’ adipocytes (from both genders) grown in 12 well plates were washed twice with DPBS +Mg2+ and Ca2 + and insulin starved in EBM-2 medium for 16 - 19 hours. 50 % confluent HDLECs were seeded on 13 mm diameter coverslips (0.16-0.19 thickness) and in the day of experiment cells were gently placed on Nunc plastic inserts (cells facing down). Importantly, the original 0.4 µm pore membrane was previously removed from the insert as it obscured the viewing of cells in microscope. After the insulin starvation of adipocytes, medium was aspirated and inserts with HDLECs were placed in the well. Cells were then gently overlayed by 1.7 ml EBM-2 medium supplemented with transferrin (0.1 ug/ml). After 24 h of incubation, HDLECs were fixed for
62 immunofluorescence staining (IF) or lysed for gene expression analysis. Adipocytes were washed once and used for a standard lipolytic assay.
5.7.2.4 Direct coculture system
To ensure proper media adaptation, fully differentiated adipocytes (standard culture, male donors) growing on coverslips were washed twice with DPBS + Mg2+ and Ca2+ and then refed with EBM-2 medium supplemented with transferrin (0.1 ug/ml). Then medium was aspirated, and HDLECs resuspended in EGM-2 medium with 2 % fetal bovine serum were added at a density 2.5 x 104 cells/cm2 directly on adipocytes to generate a direct coculture system. The cell density was chosen according to our experimental cultivation to ensure adequate cell growth. As controls, monocultures of adipocytes and HDLECs were maintained in the same way as the cells in coculture.The coculture was washed twice with DPBS + Mg2+ and Ca2+ and refed with FBS-free EGM-2 medium with or without VEGF- C (50 ng/ml) after 24 hours of incubation, which was the time required for HDLEC attachment. The medium was changed every two days. At day 2 and 7 CM was collected under aseptic conditions for theassessmentt of basal lipolysis. The cells were then fixed for detection of lymphatic and adipogenic markers by IF.