Rabbit bladder smooth muscle cell culture assay

Summary

Rabbit bladder smooth muscle cell culture can be used to (1) isolate and obtain high purity rabbit bladder smooth muscle cells, (2) perform cytological characterization studies, and (3) be used for other studies in cytology.

Operation method

enzyme separation

Principle

The smooth muscle layer of the bladder was completely isolated after carefully removing the mucosal and plasma layers of the bladder under the naked eye using microsurgical instruments. Rabbit bladder smooth muscle cells were then obtained by enzyme isolation, and the cells were isolated by primary and passaged culture in vitro. The morphology and growth of the cells were observed under an inverted microscope, and morphological observation and analysis such as H-E staining of cell crawls and transmission electron microscopy were also carried out, and immunofluorescence staining of α-actin, which is specific to smooth muscle, was applied for cytological identification and analysis. Finally, the cell growth and proliferation curves were plotted according to the cell counts.

Materials and Instruments

Male New Zealand White Rabbit
DMEM Type II Collagenase α-Smooth Muscle Actin Antibody Fetal Bovine Serum Trypsin D-Hanks' Liquid
Lunch box Ophthalmic straight scissors Ophthalmic curved scissors Ophthalmic straight tweezers Ophthalmic curved tweezers Glass Petri dishes Wide-mouth flasks Beakers Conical flasks Centrifuge tubes Magnetic stirrer Stirrer Eye nylon sieves Needle filters

Move

I. Experimental Methods

1. Enzymatic isolation of bladder smooth muscle cells
(1) Ketamine hydrochloride 200 mg and haloperidol 5 mg were injected intramuscularly to anesthetize the rabbit. After the rabbit entered the anesthesia state, it was sterilized, and an incision was made in the middle of the lower abdomen, and the bladder tissues were cut out quickly.(2) The bladder tissue was soaked in gentamicin solution (concentration of 100 units/mL), saline, and D-Hanks' solution for 5 minutes, and then put into D-Hanks' solution to remove the mucous membrane, submucosa, and plasma membrane layer.(3) The muscles were cut into minced meat and digested overnight (about 10-12 hours) in 0.1% type II collagenase (2 mg/mL) solution 40.(4) Then add 0.25% trypsin 370 digestion for 30 minutes, the digested liquid becomes turbid and flocculent suggesting good digestion.(5) Filter the flocculent solution through a 100-mesh cell sieve, centrifuge the suspension (1000 rpm, centrifugation radius 13 cm) for 5 minutes, transfer the precipitated cells to a petri dish, add DMEM containing 10% fetal bovine serum, and place it in an incubator with 50 mL/L _CO2 and 37℃ saturated humidity for static incubation, and change the incubation medium 2-3 times a week.
2. Passage culture
(1) When the cultured cells reach about 80% confluence through proliferation, do the passaging treatment.(2) Discard the old culture medium in the original culture flask, add an appropriate amount of PBS (because the fetal bovine serum in the culture medium has an inhibitory effect on trypsin), shake gently to clean the residual culture medium, and then discard it.(3) Add appropriate amount of digestive solution to cover the whole cell, place the culture flask in a _CO2 incubator and observe under an inverted microscope from time to time. When the cytoplasm of the cell retracts and the intercellular space increases, aspirate the digestive solution and add 10% fetal bovine serum to the culture flask to terminate the digestion.(4) Use a pipette to suck up the culture solution in the culture flask, and repeatedly blow the wall of the flask to prepare cell suspension, not too hard when blowing, and try not to have bubbles, so as not to damage the cells.(5) After counting the cells, inoculate the cells in the culture flask at a density of ^1X105~106 cells/mL.
3. Observation and identification of cultured cells
Inverted microscope was used to observe the morphology and growth of smooth muscle cells. The cell morphology was observed by HE staining, electron microscopy and immunohistochemical staining for α-SMA.

Results

All specimens of two rabbits were successful. Inverted microscope observation of smooth muscle cells 24 hours can be seen bladder smooth muscle cells adhering to the wall growth, normal rabbits about 7 days, while the obstruction of rabbits need 10-12 days in 50 ml culture flasks can be about 80% confluence.

All of the cells were successfully passaged, and it took about 6 days for normal rabbits and 8-9 days for obstructed rabbits to reach 80% confluence in 50 ml culture flasks after passaging.

In this group, the smooth muscle cells were passed on to the 8th generation, and after the 8th generation, the smooth muscle cells were still growing rapidly without any signs of senescence.

Under the inverted microscope, the smooth muscle cells showed "valley and peak"-like structures (Figure 1: cells in the control group at 2 weeks; Figure 2: cells in the experimental group at 2 weeks).

The HE staining of cell crawls showed that the nuclei of bladder smooth muscle cells were ovoid smooth muscle cells with bluntly rounded ends (see Figure 3).

Electron microscopy showed the smooth muscle cell dense class structure (Figure 4).

Immunohistochemical staining detected a positive reaction for α-SMA (Fig. 5).

From the HE staining of cell crawls and the positive reaction of immunohistochemical staining for α-SMA, we found that the purity of bladder smooth muscle cells obtained by this method was almost 99%.
Figure 1. Cells from the control group at 2 weeks

Figure 2. Cells of the experimental group cultured for 2 weeks.

Figure 3. HE staining of bladder smooth muscle cellswith ovoid smooth muscle cell nuclei with rounded ends.

Figure 4. Electron microscopy of cultured cells after isolation revealed dense patchy structure of smooth muscle cells
Figure 5. α-SMA immunohistochemistry confirms that this method yields a single bladder smooth muscle cell (brown color is α-SMA-positive)

Caveat

1. Strict asepsis should be practiced.

2. Careful and thorough debridement of the bladder mucosa, submucosa and plasma layer is essential to ensure that a large number of high quality individual bladder smooth muscle cells are obtained.

3. Bladder smooth muscle tissue should be miniaturized as much as possible to ensure adequate digestion.

4. Strictly control the concentration of collagenase and trypsin and the digestion time, and terminate the digestion when the digestive fluid is cloudy, the tissue mass is flocculent, or the digestive fluid has a high number of single cells or cell clumps under the inverted microscope.

5. The use of cell sieves is also the basis for obtaining high quality individual bladder smooth muscle cells.

Common Problems

I. Experimental discussion

The culture methods of smooth muscle cells can be divided into two categories: tissue block method and enzyme digestion method. Tissue block method is suitable for delicate and fragile tissues; its method is relatively simple; however, it is easy to produce impurities such as fibroblasts, etc. Fibroblasts grow fast, so the quality of the cultured cells is poor; most of the cultured cells are not contractile; and it takes 3-4 weeks to obtain primary cells, and it takes a long time to obtain a large number of smooth muscle cells ^[1]. In the past, the enzyme digestion method was more complicated and delicate than the tissue block method; it was difficult to determine the appropriate enzyme concentration and incubation time; however, a large number of smooth muscle cells could be obtained in a relatively short period of time, and Chambers et al. ^{[2] reported} that the purity of the enzyme digestion method was 70% in 1996. It can be seen that a rapid, efficient and high purity method of enzyme digestion for bladder smooth muscle culture is particularly important.

The present experimental study of all specimens from two rabbits was successful. Inverted microscope observation of smooth muscle cells 24 hours can be seen bladder smooth muscle cells adhering to the wall growth, normal rabbits about 7 days, while the obstruction of rabbits need 10 to 12 days in 50ml culture flasks about 80% confluence.

All the cells were passed on smoothly, about 6 days in normal rabbits and 8-9 days in obstructed rabbits after passaging to 80% confluence in 50 ml culture flasks.

In this group, the smooth muscle cells were passed to the 8th generation, and after the 8th generation, the smooth muscle cells were still growing rapidly without any signs of senescence.

Under the inverted microscope, the smooth muscle cells showed a "valley and peak"-like structure.

The HE staining of the cell crawls showed that the nuclei of the bladder smooth muscle cells were ovoid smooth muscle cells with bluntly rounded ends.

Electron microscopy showed that the smooth muscle cells were densely packed. Immunohistochemical staining was positive for α-SMA.

We found that the purity of the bladder smooth muscle cells obtained by this method was almost 99% from the HE staining of cell crawls and the positive reaction of immunohistochemical staining for α-SMA (this was also confirmed in the following confocal assay of bladder smooth muscle cells).

The identification of bladder smooth muscle ^{[3] is mainly} based on its cell morphology and the detection of α-actin.

Under the inverted microscope, all of them showed "valley and peak"-like structures; HE staining of cell crawls showed that the nuclei of bladder smooth muscle cells were ovoid smooth muscle cells with bluntly rounded ends; and electron microscopy showed that the smooth muscle cells were densely packed, which was confirmed by these cytomorphological tests.

Positive immunohistochemical staining for α-SMA further confirmed that the cells obtained by this method were bladder smooth muscle cells.

The post-infarcted bladder smooth muscle cells took significantly longer to grow and expand than normal bladder smooth muscle cells, suggesting that this enzymatic digestion method does not affect bladder smooth muscle cells too much and retains their essential properties in vivo.

In our laser scanning confocal microscopy, the fluorescence intensity of calcium ions in smooth muscle cells changed significantly under the influence of M receptor agonists, which further confirmed that bladder smooth muscle isolated and cultured by this method still retained its contractile function.

II. References

1. Korkmaz M, Guvenc BH, Bilir A et al. Isolation and culture of adult and fetal rabbit bladder smooth muscle cells and their interaction with biopolymers. J Pediatr Surg, 2003,38(1):21-24.

2. Chambers P,Neal DE,Gillespie JI. ^Ca2+ signalling in cultured smooth muscle cells from human bladder.Exp Physiol,1996,81(4):553-564.

3. ChamLey CJ,Campbell GR,Ross R. The smooth muscle cells in culture. Physiol Rev,1979,599(1):1-61.

4. Sui GP, Wu C, Fry CH .A description of ^Ca2+ channels in human detrusor smooth muscle. BJU Int,2003,92(4):476-478.

5. Sui GP, Wu C, Fry CH. The electrophysiological properties of cultured and freshly isolated detrusor smooth muscle cells. J Urol, 2001,165(2):627-632 .

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