Biomedical Engineering - Technology for Regenerative Medicine

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1 Whole liver regeneration A group of researchers (Uygun et al. [1]) demonstrated a novel approach to generate transplantable liver grafts using decellularized liver matrix. The decellularization process preserves the structural and functional characteristics of the native microvascular network, allowing efficient recellularization of the liver matrix with adult hepatocytes and subsequent perfusion for in vitro culture. The re-cellularised graft supports liver- specific function including albumin secretion and urea synthesis at comparable levels to normal liver in vitro. The re-cellularised liver grafts can be transplanted into rats, supporting hepatocyte survival and function with minimal ischemic damage. These results provide a proof of principle for the generation of a transplantable liver graft as a potential treatment for liver disease. Reference [1] Uygun BE, Soto-Gutierrez A, Yagi H, et al. Organ reengineering through development of a transplantable re-cellularised liver graft using decellularised liver matrix. Nat Med. 2010;16(7):814-820 2 Answer to the following questions What is the therapeutic product described above? What are the elements that identify such therapeutic product as a PTC? In what phase of the regulatory process for new PTCs can this PTC be localized? What are the standards that apply in this stage of the PTC process of development? What are the risks associated with this PTC (immunogenic, tumour, teratoma, infection, toxicity)? 3 Classify all the cell sources potentially usable in human patients for this therapy Cell source (based on immunogenicity) Cell type/s Advantages Criticalities It is usable? (Y/N) Note: CONVENTIONAL THERAPY: allogenic orthotropic transplantation 4 EXERCISE: In this exercise, the strategy described above is applied on a human liver. A. The real diameter of hepatocyte’s nucleus is D R = 5 µm; the diameter obtained from the histology, is D H = 0.1 cm. The area of histology section viewed with the microscope is A H = 0.71 x 0.87 cm 2. Considering the histology in figure 1 with a thickness tk = 2 µm, calculate the density (cells/mL) of hepatocytes leaked from the sinusoidal capillary to ECM. Figure 1: Histology of leaked cells [2] B. Considering the geometry in figure 2, evaluate the culture medium flow (Q CM ) to ensure that the wall shear stress is kept below of a critical value (τ lim = 2 Pa). The culture medium viscosity and the capillary’s diameter (from the histology in figure 1) are assumed to be 7*10 -4 Pa*s and 20 µm, respectively. Figure 2: geometry of sinusoidal capillary and tissue C. Each hepatocyte secretes P HE = 6.08*10 -12 g/h*cell of urea (CO(NH 2)2). The liver’s volume is 1500 ml. Assuming a physiological production of urea of P PH = 450 mmol/day, calculate the cell density (N v), in [10 9 cells], needed to product this amount of urea. Considering the result (N v), explain which are the implications for tissue engineering. D. Assuming that the division time of hepatocyte (t d) is 40 h, evaluate the culture time (t) in a bioreactor necessary to reach the number of hepatocytes calculated at point C (X f = 185*10 9 cells). The initial cell number is X i = 50*10 6 cells. 1 Whole liver regeneration A group of researchers (Uygun et al. [1]) demonstrated a novel approach to generate transplantable liver grafts using decellularized liver matrix. The decellularization process preserves the structural and functional characteristics of the native microvascular network, allowing efficient recellularization of the liver matrix with adult hepatocytes and subsequent perfusion for in vitro culture. The re-cellularised graft supports liver- specific function including albumin secretion and urea synthesis at comparable levels to normal liver in vitro. The re-cellularised liver grafts can be transplanted into rats, supporting hepatocyte survival and function with minimal ischemic damage. These results provide a proof of principle for the generation of a transplantable liver graft as a potential treatment for liver disease. Reference [1] Uygun BE, Soto-Gutierrez A, Yagi H, et al. Organ reengineering through development of a transplantable re-cellularised liver graft using decellularised liver matrix. Nat Med. 2010;16(7):814-820 2 Answer to the following questions What is the therapeutic product described above? Decellulari sed liver matrix cellulari sed with adult hepatocytes What are the elements that identify such therapeutic product as a PTC? The main therapeutic agent are t he cells, obtained after "non -minimal manipulation" consisting in cell isolation and manipulation, and recellularization of a decellularized organ In what phase of the regulatory process for new PTCs can this PTC be localized? Animal trial What are the standards that apply in this stage of the PTC process of development? Good Manufacturing Practice (GMP) Good Clinical Practice (GCP) What are the risks associated with this PTC (immunogenic, tumour, teratoma, infection, toxicity)? Immunological rejection : YES. We do n ot know which is the cell source Tumor formation YES, from the cell scaffold and from progenitor cells Teratoma formation YES, ma ONLY if we are using redifferentiated pluripotent cells Transmission of infections YES due to donor material (cell and scaffold) or from any manipulation Administration of toxic contaminants: YES due to donor material (cell and scaffold) or from any manipulation 3 Classify all the cell sources potentially usable in human patients for this therapy Cell source (based on immunogenicity) Cell type/s Advantages Criticalities It is usable? (Y/N) Note: CONVENTIONAL THERAPY: allogenic orthotropic transplantation Autologous hepatocytes Cells isolated from the of the patient and expanded (or oval cells differentiated in hepatocyte) Immunological Compatibility no functional cell s are available NO (no functional cells are available) Autologous iPS from somatic cells of the patient, re-differentiated in hepatocytes Cells Immunological Compatibility Limitations in re - differentiation protocols Risk of teratoma NO (teratoma risk) Syngeneic Embryonic stem cells isolated from clones of the patient itself and differentiated in hepatocytes Cells Immunological compatibility with the exception of the mitochondrial DNA. Ethical, technical and regulatory limitations for cloned cells. Limitations in re- differentiation protocols. Risk of teratoma NO (teratoma risk) Allogeneic hepatocytes isolated from a human donor and expanded (or oval cells differentiated in hepatocyte) Can be Industrialized Available (even if only from matched HLA donors) Risk of immune -reaction Risk of tumor from progenitor cell YES (cell source similar to conventional therapy)* Xenogeneic hepatocytes cells isolated from a nonhuman donor Largely available from animal livestock. Can be industrialized Risk of acute immune rejection Risk of xeno-zoonoses. Limited biological functionality Risk of tumor from progenitor cell NO (tumor and acute immune reaction) *Limitations of PTC: biological matrix (source of immunogenicity); cost-benefit balance compared to conventional therapy. 4 EXERCISE: In this exercise, the strategy described above is applied on a human liver. A. The real diameter of hepatocyte’s nucleus is D R = 5 µm; the diameter obtained from the histology, is D H = 0.1 cm. The area of histology section viewed with the microscope is A H = 0.71 x 0.87 cm 2. Considering the histology in figure 1 with a thickness tk = 2 µm, calculate the density of hepatocytes (cells/mL) leaked from the sinusoidal capillary to ECM. Figure 1: Histology of leaked cells [2] Data Diameter of hepatocyte’s nucleus D R = 5 µm Diameter of hepatocyte’s nucleus, obtained from the histology D H = 0.1 cm =0.1*10 4 µm Area of histology section viewed into the microscope A H = 0.71 cm x 0.87 cm=0.6177cm 2 Thickness tk = 2 µm Leaked hepatocytes n=9 (count them from the picture) Solution: The cell density is N v= number of cells vol Volume to be considered: Vol=area of the histological section∗ (thickness of the histological section+2∗thickness of half hepatocyte ′s nucleus) The real area of histology section (A R) is: DR:D H=A R:A H→A R= 5 μm∙0.6177 cm 2∙10 8μ m2 cm2 0. 1 cm∙10 4μ m cm =3,09∙10 5 μm 2 DR/2 DR/2 tk 5 After cellularization the cell density in the graft is: Nv= n A R�tk + D R 2 +DR 2 �= 9 cells 3,09∙10 5 μm 2∙( 2 μm+5 μm) = 0.42∙10 −5cells μm 3=0.42∙10 7cells mL B. Considering the geometry in figure 2, evaluate the culture medium flow (Q CM ) to ensure that the wall shear stress is kept below of a critical value (τ lim = 2 Pa). The culture medium viscosity and the capillary’s diameter (from the histology in figure 1) are assumed to be 7*10 -4 Pa*s and 20 µm, respectively. Figure 2: geometry of sinusoidal capillary and tissue DATA Shear stress critical value τ lim = 2 Pa Medium viscosity μ=7*10 -4 Pa*s Capillary’s diameter d= 20 µm a=d/2 SOLUTION The maximum wall shear stress can be calculated with the Haagen-Poisseuille equation (handouts pg. 120): τ rz =− 4Q μ πa 3 This shear stress has to be kept below of τ lim: τ max