Method for producing three-dimensional cell tissue
A cell tissue and cell technology, applied in the field of three-dimensional cell tissue manufacturing, can solve the problem of difficult to obtain thick tissue, and achieve the effect of rapid and simple manufacturing
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Embodiment 1
[0107] Example 1. Construction of three-dimensional cell tissue using heparin and collagen (1)
[0108] In the following examples, unless otherwise specified, collagen I was used as the collagen.
[0109] will be 3.5×10 6 Cells of normal human dermal fibroblasts (NHDF) were suspended in a mixture of 150 μL of heparin / 50 mM Tris-hydrochloric acid buffer (pH 7.4) solution and 150 μL of collagen / 50 mM Tris-hydrochloric acid buffer (pH 7.4) solution . The combination of the final concentration of heparin and collagen used is as follows figure 1 shown. The resulting suspension was seeded in a 24-well cell culture insert (Corning Inc, catalog number: 3470), and centrifuged at 400 x g for 1 minute at 10°C. Thus, a cell layer is formed on the cell culture insert. Next, Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS) was added to the cell culture inserts at CO 2 Incubator (37°C, 5% CO 2 ) for 24 hours. After culturing, the constructed tissues w...
Embodiment 2
[0111] Example 2. Construction of three-dimensional cell tissue using heparin and collagen (2)
[0112] will be 3.5×10 6 The NHDF of the cells was suspended in a mixture of 250 μL of 0.1 mg / mL heparin / 50 mM Tris-hydrochloric acid buffer solution (pH 7.4) and 250 μL of 0.1 mg / mL collagen / acetic acid solution (pH 3.7) (collagen and the final concentration of heparin were 0.05mg / mL). Samples A and B were produced as follows.
[0113] (i) Sample A
[0114] The resulting mixture was seeded into 24-well cell culture inserts and centrifuged at 400 x g for 1 min at 10°C. Thus, a cell layer is formed on the cell culture insert. Next, add DMEM containing 10% FBS to the cell culture inserts in CO 2 Incubator (37°C, 5% CO 2 ) for 24 hours. After culturing, the constructed tissues were collected and paraffin-embedded sections were made. Paraffin-embedded sections were prepared according to known methods. The prepared sections were stained with HE. HE staining was performed accord...
Embodiment 3
[0118] Example 3. Further investigation of the concentration of heparin and collagen
[0119] will be 3.5×10 6 The NHDF of the cells was suspended in a mixture of 250 μL of heparin / 50 mM Tris-HCl buffer solution (pH 7.4) and 250 μL of collagen / acetic acid solution (pH 3.7). The combination of the final concentration of heparin and collagen used is as follows image 3 shown. The resulting mixture was centrifuged at 400 x g for 1 min at room temperature to obtain a viscous mass. The obtained viscous body was suspended in DMEM containing 10% FBS. The resulting suspension was seeded into 24-well cell culture inserts and centrifuged at 400 x g for 1 min at 10°C. Thus, a cell layer is formed on the cell culture insert. Next, add DMEM containing 10% FBS to the cell culture inserts in CO 2 Incubator (37°C, 5% CO 2 ) for 24 hours. After culturing, the constructed tissues were collected and paraffin-embedded sections were made.
[0120] Paraffin-embedded sections were prepared ...
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