A Co-culture Model to Study the Effect of Kidney Fibroblast-p90RSK on Epithelial Cell Survival

Abstract

Methods for the mechanistic investigations on renal fibrosis have long been concentrated on individual type of cells, such as fibroblasts and epithelial cells. However, in recent years, growing numbers of studies have been shifting toward the role of the intercellular interactions, such as communication between tubular epithelial cells and fibroblasts. Various co-culture models have been utilized in the studies of cell-cell communication and interaction. In this chapter, we describe an innovative co-culture model employing the porous membranes for spatially partitioning the cells while allowing direct crosstalk between fibroblasts and epithelial cells in an effort of mimicking in vivo environment.

1. Introduction

Regardless of the etiology, the final common pathological manifestation of chronic kidney diseases (CKD) is renal fibrosis, which is characterized by glomerulosclerosis, interstitial fibrosis, and tubular atrophy [1]. Both interstitial fibroblasts and tubular epithelial cells play essential roles in CKD pathogenesis and progression. Interstitial fibroblasts, the major matrix-producing cells, are the primary mediators of renal fibrosis associated with progressive renal failure [2–4]. Epithelial cells, in response to chronic pathogenic cues, not only undergo apoptosis leading to kidney parenchymal destruction but also contribute to the population of active fibroblasts through a transdifferentiation process [5]. Structurally, fibroblasts reside in the renal interstitium surrounding the tubules formed by epithelial cells. This proximity facilitates interstitium-epithelial interactions that are fundamental in maintaining the integrity of the kidney structure and environment, as well as the finely regulated process of adaption to pathogenic cues [6, 7]. In this chapter, we describe an innovative co-culture model employing the porous membranes for spatially partitioning the cells while allowing direct crosstalk between fibroblasts and epithelial cells in an effort of mimicking in vivo environment.

Various co-culture models have been utilized in the studies of cell-cell communication and interaction [8]. For those to study the effects of direct cell-cell contact, different cell types are seeded in the same container subsequently or after they are mixed. After the co-culture, cell changes in behavior can be detected under microscope after immunostaining [9, 10]. For those to study the paracrine interaction of different cell types, a barrier is needed. Here we describe an innovative co-culture model to study the role of a specific gene, such as p90RSK, in cell-cell (fibroblast-epithelial) communication using primary kidney fibroblasts extracted from fibroblast-specific p90RSK transgenic mice and their littermate controls. Briefly, primary fibroblasts are seeded onto the commercially available porous membrane inserts, while epithelial cells are in the bottom chambers (Fig. 1). This model is easy and reproducible. It has the flexibility to study cell-cell communication in an on-demand manner since both cell types can be put together and separated easily. This unique model also confers the advantage of pinpointing a specific signaling pathway through inhibition experiments in individual cell chambers separately before they are put together. After the co-culture, the epithelial cells can be separated and harvested with additional treatments if necessary. Of note, to minimize the interference of fibroblast-secreted factors, we use human kidney proximal tubular epithelial cell line (HKC-8) instead of mouse epithelial cells in the study.

Fig. 1.

The schematic illustration of primary fibroblast (RSK-Tg and RSK-WT) and epithelial cell (HKC-8) co-culture. Primary kidney fibroblasts and HKC-8 cells were cultured in 100 mm dishes. After almost confluent, fibroblasts were seeded into permeable membrane inserts, and HKC cells were seeded into wells of 6-well plates. After fast overnight, combine the inserts (fibroblasts) with the wells (HKC-8 cells), and H₂O₂ can be added into the bottom chamber if necessary

2. Materials

2.1. Mice

Fibroblast-specific p90RSK transgenic mice are generated by cross-breeding p90RSK-Tg^folx and S100A4 (FSP-1)-Cre mice [11, 12]. Fibroblast-specific p90RSK transgenic mice (RSK-Tg) are those with genotype of both positive FSP-1 and p90RSK, whereas their littermates with both negative genotypes serve as controls (RSK-WT). The same gender mice are used (see Note 1).

2.2. Cell and Cell Culture Reagents

1. Human kidney proximal tubular epithelial cell line HKC-8.

2. Phosphate-buffered saline without calcium and magnesium (PBS, Corning Incorporated, NY, USA).

3. Minimum Essential Medium Eagle with glutamine (MEM, Corning Incorporated, NY, USA).

4. Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12, Life Technologies Corporation, Carlsbad, CA, USA).

5. Antibiotic-antimycotic 100x (Life Technologies Corporation, Carlsbad, CA, USA).

6. 0.05% trypsin-EDTA and 0.5 M EDTA (Life Technologies Corporation, Carlsbad, CA, USA).

7. Fibroblast complete growth media: MEM with 10% fetal bovine serum, 1% antibiotic-antimycotic.

8. HKC complete grow media: DMEM/F12 with 10% fetal bovine serum, 1% antibiotic-antimycotic.

9. Serum-free media: MEM or DMEM/F12 with 1% antibiotic-antimycotic.

10. Collagenase A, dispase II, NaCl, HEPES, and CaCl₂ (Millipore Sigma, St. Louis, MO, USA).

11. Digestion buffer: 0.1% collagenase, 2.5 U/ml dispase II, 2 mM CaCl₂, 10 mM HEPES, and 150 mM NaCl in water, filter to sterilize (see Note 2).

12. Washing buffer: 0.05 M EDTA in PBS, filter to sterile.

2.3. Other Instruments and Materials

1. Dissection board, pins, standard dissecting forceps and scissors, and razor blades (see Note 3).

2. Sterile 50 ml centrifuge tubes.

3. 70 μM cell strainer (sterile).

4. Pipettes and sterile pipette tips 10 μl, 200 μl, and 1 ml.

5. Pipet-aid and sterile serologic pipettes.

6. 100 mm cell culture dishes.

7. Sterile petri dishes.

8. Incubating orbital shaker, CO₂ incubator, microscope, and centrifuge.

9. Hemocytometer.

10. Permeable polycarbonate membrane inserts (0.4 μm pore size, 4.7cm² culture area) and 6-well cell culture plates (Corning Incorporated, NY, USA).

3. Methods

3.1. Kidney Fibroblast Isolation

1. Euthanize the WT mice following the approved protocol. Soak the mice in 70% ethanol for 5 min (see Note 4).

2. Use the sterile tools to cut the skin and open the abdomen. Take both kidneys out and save them in 10 ml sterile PBS in a tube marked with WT (see Note 5).

3. Repeat the steps 1 and 2 with RSK-Tg mice and save the kidneys in 10 ml sterile PBS in a tube marked with RSK-Tg.

4. Dispose the mice corpse properly and clean the dissection board and tools.

5. Transfer the kidneys from the tube marked with WT to a sterile Petri dish with 1 ml digestion buffer. Use the razor blade to cut the kidney tissue finely to a homogenous mince of approximately 1 mm³ pieces (see Note 6).

6. Transfer the minced kidney tissue to a 50 ml tube with 4 ml digestion buffer (see Note 7).

7. Incubate the tissue in the digestion buffer at 37 °C for 90 min at 100 rpm (see Note 8).

8. Drain the digestion buffer and the tissue through a 70 μM cell strainer to a 50 ml sterile tube. Wash the tissue and the strainer with 20 ml washing buffer and drain it into the same 50 ml tube.

9. Centrifuge the tube at 260 × g for 5 min at room temperature and discard the supernatant.

10. Add 10 ml serum-free MEM media, resuspend the pellet, and repeat step 9 once (see Note 9).

11. Add 10 ml fibroblast complete growth media to resuspend the pellet (see Note 10).

12. Plate the cells into the 100 mm cell culture dish containing fibroblast complete growth media and mark the dish with WT and incubate the cells in the CO₂ incubator at 37 °C, 5% CO₂ (see Note 11).

13. Repeat the steps 5–12 using the kidneys from the tube marked with RSK-Tg instead of WT and mark the dishes accordingly.

14. Wash and change the media the second day and then every other day. Generally, the fibroblast will be confluent within 1 week.

3.2. Culture of Fibroblasts

After isolation of primary kidney fibroblasts, some contamination of other cell types may be present in the dishes. The blood cells will be washed out in the following week. Other cells will not efficiently proliferate in the fibroblast culture media and will be diluted out from the culture.

1. Following the isolation of the fibroblasts, check the fibroblasts every day under microscope. When the cells are 80% confluent, subculture the fibroblasts as following (see Note 12).

2. Remove and discard the culture media in the dish.

3. Briefly rinse the cell with trypsin-EDTA to remove the trace of FBS in the dish (see Note 13).

4. Add 2 ml trypsin-EDTA to the dish and incubate the fibroblasts at 37 °C for 5–10 min. Check the dish under microscope every couple minutes. Add 6 ml MEM growth media in the dish when the cell layer is dispersed and the cells are released.

5. Gently pipette the suspension couple times to help the cells to be detached from the bottom and transfer the cells to a 50 ml tube.

6. Centrifuge the tube at 120 × g for 10 min, discard the supernatant and resuspend the pellet in MEM growth media, and split (1:3) into new dishes with MEM growth media.

3.3. Culture of Epithelial Cells

1. Thaw the vial of HKC cells by gentle agitation in a 37 °C water bath. Keep the cap out of the water to avoid contamination.

2. Remove the vial from the water bath as soon as the contents are thawed and spray 70% ethanol to avoid contamination.

3. Transfer the contents into a 100 mm² dish with 10 ml DMEM/F12 growth media. Shake the dish gently to mix the growth media with the contents.

4. Incubate the cells in CO₂ incubator at 37 °C and 5% CO₂. Change the growth media the second day and every 3–5 days.

5. Check the cells under the microscope every day till it is 80% confluent.

6. Remove and discard the culture media in the dish.

7. Briefly rinse the cell with trypsin-EDTA to remove the trace of FBS in the dish.

8. Add 2 ml trypsin-EDTA to the dish and incubate the HKC at 37 °C for 2–3 min. Check the dish under microscope every minute. Add 6 ml DMEM/F12 growth media in the dish when the cells are released.

9. Gently pipette the suspension couple times to help the cells to be detached from the bottom and transfer the cells to a 50 ml tube.

10. Centrifuge at 120 × g for 10 min, discard the supernatant and resuspend the pellet in DMEM/F12 growth media, and split (1:3–1:6) into new dishes with DMEM/F12 growth media (see Note 14).

3.4. Co-culture of Epithelial Cells and Fibroblasts

1. Adjust the subculture ratio of HKC so that the HKC cells and the primary fibroblasts will be 80–90% confluent and are ready for the experiment at the same day (see Note 15).

2. For the primary fibroblasts, follow the steps 2–5 of the fibroblast culture (see Subheading 3.2).

3. Centrifuge the tube at 120 × g for 10 min, discard the supernatant, resuspend the pellet in MEM growth media, and gently aspirate the suspension couple times.

4. Count the number of cells in the suspension using a hemocytometer.

5. Seed 3 × 10⁵ fibroblasts with 1 ml MEM growth media in each permeable insert and add 2 ml growth media in each bottom chamber of the 6-well plate. Mark the inserts with WT or RSK-Tg according to the fibroblasts seeded (Fig. 1).

6. For the HKC cells, follow the steps 6–9 of the epithelial cell culture (see Subheading 3.3).

7. Repeat the steps 3 and 4 using DMEM/F12 growth media instead of MEM growth media.

8. Seed 3 × 10⁵ HKC cells with 2 ml DMEM/F12 growth media in each well of the 6-well plate.

9. Incubate the fibroblasts and HKC cells in the CO₂ incubator.

10. Change the growth media of HKC cells and fibroblasts to the corresponding serum-free media in the second day.

11. In the third day, remove the media of HKC cells and add 2 ml serum-free DMEM/F12 media into each well. Remove the media of the fibroblasts (including the inserts and the bottom chambers) and add 1 ml of serum-free MEM media into each insert.

12. Transfer the inserts seeded with WT or RSK-Tg fibroblasts into different wells seeded with HKC cells immediately after step 11 using sterile forceps and mark the wells correspondingly (see Note 16).

13. Add H₂O₂ into the bottom chambers according to the experiment setting and shake gently. Incubate the plates in the CO₂ incubator for 16 h.

14. Remove the inserts, extract the protein from the bottom HKC cells, and detect different signaling (such as for cleaved-caspase 3, etc.) through Western blotting [11] (see Note 17).

4. Notes

1. We use the mice of the same age (6–10 week) and gender to eliminate the non-specific effect.

2. The collagenase A and dispase II can be filtered, aliquoted, and stored at −80 °C at the stock concentration individually (0.5% for collagenase A and 10 U/ml for dispase II in the lysis buffer with 2 mM CaCl₂, 10 mM HEPES, 150 mM NaCl). Make the fresh digestion buffer on the same day of the experiment.

3. All the instruments should be autoclaved and sterile before the experiment.

4. Clean and disinfect the operation surface and dissection board before euthanizing the mouse. Always process the mouse right after it is thoroughly soaked in 70% ethanol since the mouse body will be stiff and difficult to handle if not.

5. Though the mouse was soaked in ethanol, we recommend changing the forceps and scissors after skin cutting to avoid possible contamination. Spraying 70% ethanol to the kidneys and then washing the kidneys with sterile PBS are optional. Save the kidney in sterile PBS to prevent the tissue from drying out.

6. Step 5 and the following steps should be carried out under sterile conditions and in a cell culture hood. The entire reagent used in step 5 and following steps should be sterile. The 1 ml of digestion buffer in the Petri dish here is used to prevent the tissue from drying out during the process of kidney. More digestion buffer can be added to the petri dish if necessary.

7. Adjust the volume of digestion buffer according to the number of kidneys, generally 4 ml for one kidney. Kidneys from the same mouse or same genotype mice can be cut in the same Petri dish and transferred to the same 50 ml tube.

8. The digestion time might be longer if the tissue pieces are bigger. The shaking speed could be 100–150 rpm depending on the volume of the digestion buffer.

9. Repeating the steps 9 and 10 for additional wash is optional and is recommended if fibroblasts from more kidneys are collected at the same time and saved in the same tube.

10. The volume of growth media can be adjusted according to the number of kidneys used.

11. Fibroblasts isolated from one kidney will be plated into two 100 mm plates. Adjust the volume of growth media got from step 11 and the volume of growth media pre-added in the 100 mm dishes and make sure enough fibroblasts are added into 10 ml growth media in each plate.

12. Freshly prepared fibroblasts are not good for experiment due to harsh treatment during the isolation and the possible contamination of other cell type. We recommend using the fibroblasts less than four passages as they may lose the ability of proliferation.

13. Serum may contain trypsin inhibitor.

14. We recommend using the HKC after two passages so the cells are recovered and behave normally.

15. In other experimental settings, such as pretreatment of HKC or fibroblasts is required; the subculture ratio of HKC will be adjusted accordingly so that the cells can be seeded into the inserts or 6-well plates 1–2 days in advance for the treatment before the co-culture.

16. Handle the inserts gently to avoid any media spills.

17. Since we study the effects of fibroblasts p90RSK on the epithelial apoptosis, we use H₂O₂ as apoptosis inducer of HKC cells and the cleaved-caspase 3 as a marker of apoptosis. The media can also be saved for other signaling detection if needed.