New born child tissue engineering bladder piece and preparation method thereof
A tissue engineering and neonatal technology, applied in bone/connective tissue cells, artificial cell constructs, medical science, etc., can solve the problems of cell aging, death, and inability to adapt to bladder tumor patients, and achieve obvious tissue thickening, cell Rapid proliferation and good cell attachment ability
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Embodiment 1
[0035] 1. Isolation, sorting and induction culture of hAFCSCs:
[0036] Amniotic fluid samples come from pregnant women undergoing prenatal diagnosis in the second trimester (4-6 months). Ultrasound-guided amniocentesis is used to obtain 20-40ml amniotic fluid into a 50ml sterile centrifuge tube, which is sealed and stored in a 4°C refrigerator and quickly transferred to a laminar flow sterile tube. The ultra-clean bench in the cell culture room. After filtering through a sterile 100-mesh sieve, centrifuge at 1000 rpm for 5 min at 4°C. After centrifugation, the supernatant was discarded, and 5ml of culture medium was added to the tube for cultivation. After 8-10 days, clones with good growth and uniform morphology were picked with the cloning cup to obtain hAFCSCs. After continuing to culture and expand in vitro, select the 4th passage hAFCSCs. When they grow to 70-80% confluence, replace with the basic induction medium and add induction factors to the culture dish every 24 ...
Embodiment 2
[0047] Cell fluorescent immunostaining was performed on the induced cells obtained in Example 1, and it was confirmed that hAFCSCs induced by the above induction scheme expressed smooth muscle cell marker smooth muscle actin (α-smooth muscle actin, α-SMA) ( figure 2 A), troponin (calponin, CALP) ( figure 2 B) and myosin heavy chain (MHC) ( figure 2 C), demonstrating that our induction method can successfully differentiate hAFCSCs into smooth muscle-like cells.
[0048] Immunofluorescent staining of tissue immunofluorescent staining found that smooth muscle cell marker protein α-SMA, CALP, and MHC all expressed ( image 3 arrow pointing).
[0049] The PGA material support and compound that obtain in example 1 two, three are carried out scanning electron microscope observation and visible non-woven PGA support material is irregular grid-like structure ( figure 2 A), the grid size is conducive to the penetration and migration of hAFCSCs into the scaffold; at one week, it c...
Embodiment 3
[0050] Example 3 Evaluation of the mechanical effect of the composite of hAFCSCs planted in vivo and PGA material scaffold:
[0051] Trim the sample obtained in Example 14 into a rectangular shape with a size of about 15mm×10mm×1.5mm. The specimen is fixed on a universal electronic stretching machine (instron 5969), using a 50N tension sensor at a speed of 5mm / min. Load the sample, detect the maximum tensile stress (that is, the maximum load), the maximum elongation rate, the deformation at the maximum load and other indicators, and the measured data are analyzed and processed by the statistical software SPSS.11.0, and the mechanical indicators of each group Expressed as mean ± standard deviation (Table 1). The comparison of the mechanical data between the samples in each group was performed by the Student’s t test, and P Figure 4A ); the mechanical experiment group was compared with the mechanical positive control group (P=0.0103), and the difference was significant ( Figure ...
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