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Biomedical Engineering - Technology for Regenerative Medicine

ese06-ENG

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1 Stomach regeneration: Murine Tissue-Engineered Stomach Demonstrates Epithelial Differentiation 1) Speer et al. harvested neonatal stomach epithelial cells and seeded a polyglycolic acid (PGA) based porous scaffold. The seeded scaffold is implanted in adult mice for one week. Eventually cellularisation was evaluated using histology. Answer to the question in the table: 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)? 2 Classify all the cell sources potentially usable in human patients for this therapy Cell source (based on immunogenicity) Cell type/s Advantages Criticalities 3 2) The polyglycolic acid (PGA) is a scaffold with a porosity equal to 95%. It was cellularized with a cellular density of (0.019±0.007)* 10 6������������������������������������������������������������ ������������������������ 3. The scaffold could be modeled as a hollow cylinder with a height of 3mm (H=3mm), internal radius of 2.25mm (R inC) and external radius of 4.25 mm (R outC ). After the seeding and before the implantation, researchers had to store up the seeded scaffold in (Vol medium ) 5ml standard medium, continuously stirred. How much time, the scaffold could be kept in the stirred medium without causing hypoxia? After calculations plot the oxygen concentration profile. Also consider these data: - V max (the worst case for oxygen consumption due to the maximum cell density N i(max) ); - c 0 = 0.2 ������������������������������������������������ ������������������������ (initial concentration) - K m = 0.15 ������������������������������������������������ ������������������������ (constant saturation) - Cellular oxygen consumption: V cell = 0.18 ������������������������������������������������ ������������∗������������������������ ������������������������������������������������������������������������ - Hypoxic condition: c = 0.1*c 0 - Vol medium = 5ml 4 3) Once implanted, the scaffold takes a spherical configuration: assume that sphere has an internal radius of 1.13 mm (R inS) ) and the thickness is 2mm (R outS = R inS + 2 mm = 3.13 mm). In this way, the volume of the sphere and the cylinder are the same. Assume venous pO 2 both at the inner and at the outer wall (pO 2 = 40 mmHg). Assume D O2 inside the scaffold equal to 8*10 -5 ������������������������ ������������ ������������ . Calculate concentration profile c(r) assuming stationary conditions and spherical symmetry. Calculate the region of the scaffold in which the oxygen concentration is above the critical value (c(r) > 0.1*c0) and the fraction of the scaffold that is oxygenated. 5 4) From 1-week histology it is possible to estimate a cellular density of 0.79 106cells mm 3 Calculate the number of cells initially present in the volume of the scaffold where the condition c(r) > 0.1c 0 is satisfied (V o2). Assume a doubling time (t d) of 24 hours. Using these data, calculate the theoretical final cells number. Compare the final cellular density (using V o2), with the estimated one. Try to explain the results obtained. What do you think it could be modified, to reach a more performing regeneration system? 1 Stomach regeneration: Murine Tissue -Engineered Stomach Demonstrates Epithelial Differentiation 1) Speer et al. harvested neonatal stomach epithelial cells and seeded a polyglycolic acid (PGA) based porous scaffold . The seeded scaffold is implanted in adult mice for one week. Eventually cellularisation was evaluated using histology. Answer to the question in the table : What is the therapeutic product described above? PGA porous scaffold cellularized with epithelial cells 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 expansion 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 , the cell source is not autologous (recipient = adult mice) . Tumor formation YES, from the cell scaffold and from niches of stem cells existing in the stomach tissue . Terato ma formation NO , unless we are using pluripotent cells re- differentiated in epithelial cells . Transmission of infections YES , from donor material or from any manipulation . Administration of toxic contaminants : YES , due to the scaffold degradation and from any manipulation . 2 Classify all the cell sources potentially usable in human patients for this therapy Cell source (based on immunogenic ity) Cell type/s Advantages Criticalities Autologous Epithelial Cells isolated from the stomach of the patient (and expanded ) Immunological Compatibility Limited availability and expandibility Invasive procedure Autologous iPS from somatic cells of the patient, re-differentiated (in Epithelial Cells ) Immunological Compatibility Limitations in re -differentiation protocols Risk of teratoma Syngeneic Embryonic stem cells isolated from clones of the patient itself and diffe rentiated (in Epithelial Cells ) Immunological compatibility except for the mitochondrial DNA. Ethical, technical and regulatory limitations for cloned cells Limitations in differentiation protocols. Risk of teratoma from stem cells Allogeneic Epithelial cells isolated from a human donor (and expanded ) Can be Industrialized Fairly available, considering only the use of only human leukocyte antigen matching (HLA) cells Risk of immune -reaction Risk of tumor from stem cells existing in the stomach tissue Xenogeneic Epithelial cells isolated from a non - human 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 stem cells existing in the stomach tissue 3 2) The polyglycolic acid (PGA) is a scaffold with a porosity equal to 95%. It was cellularized with a cellular density of (0.019±0.007)* 10 6����� �� 3. The scaffold could be modeled as a hollow cylinder with a height of 3mm (H=3mm ), internal radius of 2.25mm ( RinC) and external radius of 4.25 mm ( RoutC ). After the seeding and before the implantation, researchers had to store up the seeded scaffold in (V ol medium ) 5ml standard medium, continuously stirred. How much time, the scaffold could be kept in the stirred medium without causing hypoxia? After calculations plot the oxygen concentration profile. Also consider these data: - Vmax (the worst case for oxygen consumption due to the maximum cell density Ni(max) ); - c0 = 0.2 ��������� �� (initial concentration) - Km = 0.15 ��������� �� (constant saturation) - Cellular oxygen consumption: V cell = 0.18 �������������� ������∗���������������������������� - Hypoxic condition : c = 0.1*c 0 - Vol medium = 5ml Data: - Scaffold porosity: 95% - Worst cellular density : Ni= Ni(max) = (0.019+ 0.007 ) 10 6����� �� 3 = 0.026 *10 6����� �� 3. - Scaffold height: H = 3 mm - Internal scaffold radius: R inC = 2.25 mm - External scaffold radius: R outC = 4.25 mm - Vmax (the worst case for oxygen consumption due to the maximum cell density Ni(max) ) - c0 = 0.2 µmol/ml (initial concentration) - Km= 0.15 µmol/ml ( constant saturation) - Cellular oxygen consumption: V cell = 0.18 ��������� ℎ∗106����� ; - Hypoxic condition : c = 0.1*c 0 - Vol medium = 5ml SOLUTION: Calculate the oxygen concentration reduction in the control volume, by using the compar tmental model of mass transport : �� �� ∗�������� ����������� = ���������������� − ���� ���� + �− � where : Qin and Q out are the flow rate s, which flow in and out in the control volume ( Qin = Q out =0), p is the oxygen produced ( p = 0) � is the oxygen consumption in the control volume (the scaffold): �= ������ ∗�������� �������� 4 Considering the simplified assumptions, the resulting equation is : �� �� ∗�������� ����������� = −������ ∗�������� �������� where V olmedium is the volume of medium and the consumption V has a different expression depending on oxygen concentration (Michaelis -Menten kinetics) . ������ = �������������� ∗ � �� + � a) If c ≥ K m, V=V max b) If c < K m, V=( ������������������� �� )* c Where V max has the following expression: �������������� = ������������ ���������� (1− ������) ε is the non -consuming fraction . In this case, since the porosity is 95% , the non -consuming fraction is 5%. Therefore, 1-ε corresponds to 0.95. Vcell is the cellular oxygen consumption = 0.18 µmol 106 ����� ∗ℎ The worst case for oxygen consumption occurs when the cell density is maximum: Ni= 0.026 *10 6����� �� 3 [NB: 1ml= 1000 mm 3] Therefore, �������������� = ���������������������� (1− ������)= 0.026 10 6 ����� �� 30.18 µ��� 10 6 ����� ∗ℎ 0.95 = 0.004446 µ��� �� 3∗ℎ 10 3�� 3 �� ℎ 3600 �= 0.00123 µ��� �� ∗� Volscaffold is the scaffold (a n empty cylinder) volume, and its expression is: �������� �������� = (����� 2− ��������� 2)������∗������ = (18 .06 − 5.06 )������∗3 = 39 ������ �� 3 = 122 .5 �� 3 �� 10 3�� 3= 0.1225 �� Calculate the total time ( or storage time, ts) as �� = ���>������� + ���������� Therefore, �� �� ∗�������� ����������� = −�������������� ∗�������� �������� W e are interested in finding the time ���>������� at which c = Km. �� = −�������������� ∗�������� �������� �������� ����������� ∗�� 5 Performing integration by variable separation c(t) expression results: �(�)= −�������������� ∗�������� �������� �������� ����������� ∗�+ � Applying the initial condition c = c0 at t = 0 �(0)= �0= −�������������� ∗�������� �������� �������� ����������� ∗0+ � = � A = c0. �(�)= −�������������� ∗�������� �������� �������� ����������� ∗�+ �0 The time � at which c = Km is: �(���>�� )= ������� = −�������������� ∗�������� �������� �������� ����������� ∗���>�� + �0 ���>�� = (�0− ������� ) �������������� ∗�������� �������� ∗�������� ����������� = (0.20 − 0.15 )µ��� �� ∗5�� 0.00123 µ��� �� ∗� 0.1225 �� ≅ 1659 �≅ 27 �������� b. For c < Km, The concentration profile decreases from the value K m to c min V = (������������������� �� )*c C = Km at t’ = 0 (NB: to simplify the calculation, it is appropriate to consider the initial time equal to 0 again). Calculate ��� 0.1c 0 is satisfied (V o2). Assume a doubling time (t d) of 24 hou rs . Using these data, calculate the theoretical final cells number. Compare the final cellular density (using V o2), with the estimated one. Try to explain the results obtained. What do you think it could be modified, to reach a more performing regeneration system? DATA: - Cellular density : NHysto = 0.79 10 6cells mm 3 - VolO2 =10.21 mm 3 - td = 24 - Average density previously declared: ������������= 0.019 106cells mm 3 SOLUTION : The initial average number of the cell seeded in V olO2 of the scaffold is : ������������= ������������∗�������� ������2= 0.019 10 6cells mm 3 ∗10 .21 �� 3= 0.19 x10 6cells Knowing the doubling time (t d = 24 hours ), the theor etical final cell number after 1 weeks will be: T = td * d = t d ln(�������������������) ��2 ln (������� ������������)= ��2 � �� �������= ������������∗2 ���= 0.19 ∗10 6cells ∗2 16824 = 24 .32 ∗ 10 6cells �������= ������� �������� ������2 = 24 .32 ∗10 6cells 10 .21 �� 3 = 2.38 10 6cells mm 3 It is possible to notice that theoretic N f is different from the estimated one (Nhysto =0.79*10 6cells/mm 3). A first consideration is that cells in hypoxic condition send p aracrin signals influenc ing cell survival in cells found in the scaffold region with sufficient oxygenation. For this reason , in the future, it is mandatory to change the geometry of the scaffold so that there is a high fraction of oxygenated cells. A possible further explanation ca n be added by evaluating whether within a week there is the negative influence of the degradation products of the scaffold that could negatively interact with cellular metabolism causing their death.