Isolation and culture of adult and fetal rabbit bladder smooth muscle cells and their interaction with biopolymers☆

Materials and methods 

Ten young adult New Zealand white rabbits and 12 fetuses at 25 days' gestational age were used as the tissue donors. Adult and time-mated pregnant rabbits were anesthetized with intramuscular xylazine, 5 mg/kg, and ketamine, 25 mg/kg. After laparotomy, bladder tissues approximately 2 cm2 in size were obtained by partial resection of the bladder in the adult, whereas the whole bladder was resected in fetus. The specimens were processed within one hour after surgical removal.

The cells were cultured in Dulbecco's modified eagle medium (DMEM), supplemented with 10% fetal calf serum (Sigma Chemical Co, St Louis, MO), 2 mmol/L L-glutamine, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. The bladder wall was stripped of its mucosa and placed in culture medium; detrusor smooth muscle was cut into small pieces with scissors and lancets under sterile conditions. The suspension was placed onto dishes for explant culture, and 10 mL medium was added to each 100-mm dish. Cells were grown at 37°C in an incubator with a humidified atmosphere of 5% CO2 in air.

Culture medium was changed twice weekly. When cultures reached a confluence of about 80%, cells were resuspended by incubating them with trypsin (Serva, 0.025%) and EDTA (Serva, 0.2%). They were maintained and expanded with the same culture medium.

Results 

Cell cultures obtained from fetal tissue were seen to emerge from the explants after 3 to 4 days, whereas adult cells emerged after 5 to 6 days. First cell passage was performed after 12 to 14 days when the cell cultures reached confluence. Light and SEM findings of all SMCs displayed characteristic spindle-shaped morphology and centrally located round to oval nucleus. At low density, multiple pseudopodia emanated from the cells until they made contact with adjacent cells, whereupon becoming classic spindle-shaped and forming a hill-and-valley appearance of the culture was observed (data not shown).

Immunocytochemistry showed that over 90% of the adult and fetal cell populations stained positive for SMC α-actin and myosin. Fetus and adult cell populations at 48th and 96th hour are shown in Fig 1.

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Fig. 1. Fetal and adult SMCs numbers at 48th and 96th hours.

Cells were seeded on FCS or fibronectin-adsorbed biopolymers, and after a 7-day incubation, their numbers were determined by the neutral red uptake assay (Fig 2).

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Fig. 2. Cell growth experiments performed by the neutral red uptake assay. The indicated polymers were incubated for 1 hour with 1 mg/mL fibronectin or 10% FCS containing medium before seeding with (A) 48,000 per well adult, (B) 69,000 per well fetus SMCs. The assay was performed after 7 days in culture.

Discussion 

The improvements of selective cell transplantation have provided hope of a means of formation of a functional bladder wall. Autologous tissue replacement has potential anatomic and functional advantages. Engineering of tissue using autologous cells, already has been applied in various clinical settings for a variety of disorders including the use of engineered skin for burn patients, urothelium for repair of hypospadias, cartilage for knee replacement, and injectable chondrocytes for treatment of vesicoureteral reflux.10, 11, 12

Several groups have studied the growth of urothelium in vitro, and the physiologic and histologic characteristics are well defined. There are a few studies on bladder smooth muscle cell properties such as extracellular matrix proteins and in vitro contractility testing.13, 14 However, after bladder regeneration with matrices, histologic investigations showed that urothelial regeneration was rapid, but muscular layer was absent or poorly developed.15 Smooth muscle layer is important to achieve a functional bladder wall. In this study we aimed to investigate and compare the growth properties of SMCs derived from the fetal and the adult rabbit bladder in vitro. We also studied their growth rates on commonly utilized biopolymers.

Tissue specimens may be processed by enzymatic digestion or mechanical disruption (tissue explant technique) methods before seeding onto dishes. We showed that cutting tissues into small fragments before seeding is feasible and cheap because all explants were cultured easily. Light and SEM images showed that there were no morphologic differences between fetus and adult cells.

After tissue explanation, fetal cells emerged from tissue earlier than adult cells, but their PDT and TLI values at second passage of culture showed that they were proliferating significantly slower than adult cells. SMCs display extensive plasticity of phenotype; this is especially true in vascular tissue16 where most studies have been performed because of its relevance to atherosclerosis. During in vivo rat aortic development, SMC proliferation has been reported to decrease from 80% per day (until embryonic day 18) in skin cells17 to an adult rate of less than 0.006% per day in bladder SMCs.18 Our results are contrary to the above findings. In our in vitro study, the initial outgrowth of fetal cells from the explants are faster, but 2 passages made in the 10% FCS containing cell culture medium may have adversely affected the proliferation rate of fetus cells.

Contamination with other cells such as stromal cells still is a common problem experienced by other observers. In this study, we have shown that cultures grew with low stromal contamination and were stained over 90% positive with SMCs marker protein antibodies. We selected high glucose and calcium concentration type of DMEM. It seems that meticulous dissection of tissue and appropriate culture medium chosen for cell culture are very important to achieve selective cell strains.

Numerous studies have been made with PLGA biopolymers, because these materials are designed as biocompatible, biodegradable, and may be modified to improve cell growth and proliferation. In some studies with human bladder SMCs, PLGA with a 90:10 monomer ratio has been used.7 In this study, adult and fetus SMCs were seeded on 75:25 and 85:15 PLGA biopolymers adsorbed with proteins and their ability to support cell attachment and proliferation were compared. Because of its highly hydrophobic nature, 85:15 PLGA enabled less cell growth than the more hydrophilic 75:25 PLGA irrespective of the type of protein adsorbed. This finding is in parallel with reports indicating that moderate surface wettability is essential for optimal cell attachment onto polymer surfaces.19 As other cells, SMCs interact with extracellular matrix proteins such as fibronectin and vitronectin through a group of heterodimeric cell membrane proteins called integrins.20 Our results indicate that adsorption of FCS, which contains 0.25 to 0.45 mg · ml−1 vitronectin, has resulted in better cell proliferation compared with fibronectin-adsorbed polymers. These results show the importance of protein adsorption as a means of surface modification of polymeric materials.