Cardiac or vascular tissue spheroid
A heart tissue and tissue-type technology, applied in cardiovascular system diseases, tissue regeneration, vascular endothelial cells, etc., can solve problems such as morphological characteristics or mechanical action mode technology has not been found.
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Embodiment 1
[0092] (1) Preparation of cells
[0093] (1-1) Preparation of primary cardiomyocytes (rat fetal ventricular cardiomyocytes) (CM) (preplating method)
[0094] The SD (kud: sd) neonatal rats on day 1 to 3 after birth were decapitated, the chest was opened and the heart was removed.
[0095] Wash 3 times with HBSS+ / + (Hank's Balanced Salt Solution (Hank's Balanced Salt Solution), containing Mg, Ca: sigma aldrich) cooled to 4°C, and then wash with HBSS- / - (excluding Mg, Ca) 3 times.
[0096] After removing the atria and great blood vessels so that only the ventricle remained, the ventricle was finely cut with scissors to obtain fragments with a size of 1 mm or less. The finely cut ventricular fragments were placed in 0.1% trypsin solution (Wako Pure Chemical Industries, Ltd.) inHBSS- / -, and left to stand at 4°C for 6 hours.
[0097] Next, 10% FBS (sigma) and DMEM (Low Glucose) containing P / S solution (Wako Pure Chemical Industries, Ltd.) were added to stop the trypsin reaction....
Embodiment 2
[0165] This embodiment is an example of preparing a three-dimensional structure by fusing spheres.
[0166] Spheroid formation of each cell was carried out in the same manner as in Example 1.
[0167] Cardiac tissue spheroids were formed by staining cardiomyocytes with a green fluorescent dye, staining vascular endothelial cells with a red fluorescent dye, and staining fibroblasts with a blue fluorescent dye. Further, the four spheres were fused, and observed after 3 hours, 12 hours, and 24 hours.
[0168] As a result, a vascular network is formed through cooperation of the three types of cells, and at the same time, a beating cardiac tissue-type sphere can be prepared ( Figure 9 ). Such as Figure 9 As shown, it was found that the three types of cells were fused. In addition, the cells undergo some sort of reprogramming, forming a new network of blood vessels.
[0169] When the spheres are fused to each other, the spheres beat in sync.
Embodiment 3
[0171] In this example, various types of spheroids were formed using one type of cells, and a heart tissue formation test was performed when each spheroid was blended.
[0172] Spheroid formation of each cell was carried out in the same manner as in Example 1.
[0173] Three types of spheres formed solely by cardiomyocytes (green), spheroids formed by vascular endothelial cells alone (red), and spheroids formed by fibroblasts alone (blue) were observed after 24 hours.
[0174] show the result in Figure 10 . Such as Figure 10 As shown, three kinds of spheres can be fused.
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