Process for producing a tissue transplant construct for reconstructing a human or animal organ
A process for producing a tissue transplant construct for reconstructing a human or animal organ. The process may include the steps of: (a) isolation and two-dimensional cultivation of organ-specific tissue cells; (b) application of the organ-specific tissue cells to a biocompatible, collagen-containing membrane; and, (c) cultivation of the organ-specific tissue cells on the membrane with biochemical and mechanical stimulation of the organ-specific tissue cells. Tissue transplant constructs and methods for using tissue transplant constructs are also taught.
1. A process for fabricating a urinary bladder tissue, comprising the steps of:
providing a collagen based acellular tissue membrane obtained from one or more of urinary bladder, urinary bladder submucosa, aorta, urethra, small intestinal submucosa or dermal matrix, the acellular tissue membrane having a first side and an opposing second side;
collecting smooth muscle cells and interstitial fibroblastic cells from a urinary bladder;
collecting urothelial cells from a urinary bladder;
culturing smooth muscle cells and interstitial fibroblastic cells in two dimensions between two to four passages to confluence;
culturing the urothelial cells in two dimensions to confluence in a plurality of passages;
seeding the first side of the acellular tissue membrane with smooth muscle cells and interstitial fibroblastic cells from a selected passage;
seeding the second side of the acellular tissue membrane with urothelial cells from a selected passage; and
culturing the seeded acellular tissue membrane by simultaneous mechanical and biochemical stimulation; wherein
the mechanical stimulation comprises applying tension cyclically to the seeded acellular tissue membrane, wherein the periodicity of the tension comprises a stretching period of about 10 seconds to about 20 seconds and a relaxing period of about 10 seconds, and the strain resulting therefrom is approximately 10%, and the mechanical stimulation continues until the acellular tissue membrane comprises confluently cultured cells; and
the biochemical stimulation includes culturing the seeded acellular tissue membrane in a serum-conditioned culture medium, wherein the serum-conditioned culture medium comprises autologous serum or fetal bovine serum conditioned by proliferating urothelial cells, and wherein the serum-conditioned culture medium comprises about 5% serum for a first cultivation period, 1% serum for a second cultivation period, and about 0% serum for a third cultivation period; and
resulting in a urinary bladder tissue comprising:
at least one layer of smooth muscle cells and interstitial fibroblastic cells, wherein said layer comprises between 75% and 85% smooth muscle cells and between 15% and 25% fibroblastic cells; and
at least one layer of urothelial cells bounded by the acellular tissue membrane.
2. The process of claim 1 , wherein the first cultivation period is about 14 days, the second cultivation period is about 13 days, and the third cultivation period is about three days.
3. A fabricated urinary bladder tissue made by the process of claim 2 .