Different clinical conditions can require urinary bladder augmentation or replacement. Tissue engineered bladder has been clinically evaluated but is not recommended due to diverse side effects. Thus, there is a real interest for the development of regenerative approach with innovative scaffolds and cell transplantation. The investigators propose the use of urothelial cells obtained by Trans-Urethral Resection of Prostate or bladder (TURP) to obtain a tissue engineered urothelium in association with different scaffolds.
Bladder biopsies will be obtained during cystoscopy, conserved in culture medium (DMEM®), digested by dispase and sowed on collagen-coated culture support. Keratinocyte Serum Free Medium (KSFM) will be used for proliferation. Microscopy, immunohistochemistry, RNA extraction, Reverse Transcription and quantitative Polymerase Chain Reaction (RT-qPCR) will be performed during passages. Cell culture conditions will be optimized to improve proliferation and avoid loss of differentiation. The investigators will develop scaffolds based on sodium alginate hydrogels, followed by freeze-drying to generate porous sponges (at -20°C and -80°C). Cultured cells will be associated to these original scaffolds and to other scaffolds, for example alginate hydrogels or Collagen Cell Carrier (CCC), cultivated for 28 days and analyzed. Histological and immunohistological appearance of cellularized scaffolds will be compared to assess the effectiveness of each scaffold for tissue engineering in urothelium. Cellularized scaffolds will be studied in vitro (Transepithelial Electrical Resistance, impermeability, ability to be stitched, resistance to urine) and in vivo in ectopic location (subcutaneous location in Nude mice) or in orthotopic location (bladder augmentation in small animal).
Study Type
OBSERVATIONAL
Enrollment
365
Transurethral resection of the prostate is a urological operation used to treat benign prostatic hyperplasia (BPH). It is performed by visualising the prostate through the urethra and removing tissue by electrocautery or sharp dissection with a resectoscope. This is considered the most effective treatment for BPH. This procedure is done with spinal or general anaesthetic. A triple lumen catheter is inserted through the urethra to irrigate and drain the bladder after the surgical procedure is complete. Outcome is considered excellent for 80-90% of BPH patients.
Histological analysis of biopsy.
Histological analysis with standard coloration will assess the viability of urothelium. Signs of necrosis (ulcerations, destructions of urothelium structure) will be noted.
Time frame: 6 month
Immunohistological analysis of biopsy.
Immunohistological analysis using specific antibodies will locate the urothelium (Keratin, Uroplakin), the smooth muscle (Smooth muscle actin), the prostatic gland (Prostatic specific antigen).
Time frame: 6 month
RT-qPCR analysis of biopsy.
RT-qPCR analysis will measure the level of expression (compared to housekeeping gene RPLP0) of RNA specific to urothelium (Keratin, Uroplakin), the smooth muscle (Smooth muscle actin), the prostatic gland (Prostatic specific antigen). This level will be compared to cultured urothelial cells.
Time frame: 6 month
Digestion of the biopsy and culture with adapted medium. Optimization of culture conditions.
The biopsy will be digested using dispase. Cells will be sowed on collagen coated supports and cultured using Keratinocyte Serum Free Medium (KSFM). Trypsination will be done at confluence. RT-qPCR (level of expression of each specific marker) will be realized at each passage to assess evolution during culture.
Time frame: 12 months
Histological analysis of the cultured cells.
Cultured cells (Outcome 4) will be analyzed in contrast phase microscopy and with standard microscopy coloration to assess their morphology, the number of cellular types and their evolution throughout passages.
Time frame: 12 months
Immunocytological analysis of the cultured cells.
Cultured cells (Outcome 4) will be analyzed in immunocytology to assess the type of cells (urothelial / smooth muscle / prostatic), the expression and the localization of specific markers (Outcome 2)
Time frame: 12 months
RT-qPCR analysis of the cultured cells.
Cultured cells (Outcome 4) will be analyzed in RT-qPCR at each passage to assess evolution during culture. The level of expression (compared to housekeeping gene RPLP0) of RNA specific to urothelium (Keratin, Uroplakin), the smooth muscle (Smooth muscle actin), the prostatic gland (Prostatic specific antigen) will be measured.
Time frame: 12 months
Development of original alginate freezed-dried scaffold
Development of an original scaffold based on sodium alginate hydrogels, followed by freeze-drying to generate porous sponges (at -20°C and -80°C). Analysis of structure, impermeability, ability to be stitched..
Time frame: 12 months
Association of the cultured cells with different scaffolds. Culture of cellularized scaffolds.
Previously cultured and analyzed cells will be associated to different scaffolds. * Collagen scaffolds * Alginate scaffolds * Alginate hydrogels obtained by spray * Original alginate freezed-dried scaffold (Outcome 4) Cellularized scaffolds will be cultured at least 28 days using previously described conditions.
Time frame: 36 months
Histological analysis of cellularized scaffolds
After association, cellularized scaffolds (Outcome 9) will be cultured at least 28 days. Survival rate using MTT assay will be performed. Histological analysis using standard colorations will be performed to evaluate the appearance of the cellularized scaffold, the location, the appearance and the organization of the cells.
Time frame: 36 months
Immunohistological analysis of cellularized scaffolds
After association, cellularized scaffolds (Outcome 9) will be cultured at least 28 days. Immunohistological analysis will be performed to evaluate the expression and location of specific markers (Outcome 2)
Time frame: 36 months
RT-qPCR analysis of cellularized scaffolds
After association, cellularized scaffolds (Outcome 9) will be cultured at least 28 days. RT-qPCR will be performed to assess evolution during culture in 3 dimensional conditions. The level of expression (compared to housekeeping gene RPLP0) of RNA specific to urothelium (Keratin, Uroplakin), the smooth muscle (Smooth muscle actin), the prostatic gland (Prostatic specific antigen) will be measured.
Time frame: 36 months
Biophysical analysis of cellularized scaffolds
After association, cellularized scaffolds (Outcome 9) will be cultured at least 28 days. Biophysical analysis on cellularized scaffolds will be done: * Transepithelial Electrical Resistance (cellular ability to form an impermeable barrier) * Impermeability (ability of the cellularized scaffold to form a water resistant barrier) * Ability to be stitched (ability of the cellularized scaffold to be manipulated and stitched) * Resistance to urine (ability of the cellularized scaffold to resist to chemical aggression of urine)
Time frame: 36 months
Implantation of the cellularized scaffold in Nude mice
Cellularized scaffolds will be implanted in subcutaneous location in Nude mice. Incubation in vivo will be done during at least 28 days. Analysis (histology, immunohistology, RT-qPCR) will be performed after 28 days of incubation to assess the survival of cells and the behavior of the cellularized scaffold in vivo. Signs of necrosis, neoangiogenesis and inflammation will be noted.
Time frame: 12 months
Histological analysis of implanted scaffolds
Histological analysis will be performed after 28 days of incubation on implanted scaffolds (Outcome 14) to evaluate the appearance of the cellularized scaffold, the location, the appearance and the organization of the cells. Survival rate using MTT assay will be performed. Signs of fibrosis will be noted.
Time frame: 12 months
Immunohistological analysis of implanted scaffolds
Histological analysis will be performed after 28 days of incubation on implanted scaffolds (Outcome 14) to evaluate Immunohistological analysis will be performed to evaluate the expression and location of specific markers (Outcome 2)
Time frame: 12 months
RT-qPCR analysis of implanted scaffolds
RT-qPCR analysis will be performed after 28 days of incubation on implanted scaffolds (Outcome 14) to assess evolution during in vivo conditions. The level of expression (compared to housekeeping gene RPLP0) of RNA specific to urothelium (Keratin, Uroplakin), the smooth muscle (Smooth muscle actin), the prostatic gland (Prostatic specific antigen) will be measured.
Time frame: 12 months
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