Measurement of the Adipose Stem Cells Cell Sheets Transmittance

Abstract

In the field of cell therapy, the interest in cell sheet technology is increasing. To determine the cell sheet harvesting time requires experience and practice, and different factors could change the harvesting time (variability among donors and culture media, between cell culture dishes, initial cell seeding density). We have developed a device that can measure the transmittance of the multilayer cell sheets, using a light emitting diode and a light detector, to estimate the harvesting time. The transmittance of the adipose stromal cells cell sheets (ASCCS) was measured every other day as soon as the cells were confluent, up to 12 days. The ASCCS, from three different initial seeding densities, were harvested at 8, 10, and 12 days after seeding. Real-time PCR and immunostaining confirmed the expression of specific cell markers (CD29, CD73, CD90, CD105, HLA-A, HLA-DR), but less than the isolated adipose stromal cells. The number of cells per cell sheets, the average thickness per cell sheet, and the corresponding transmittance showed no correlation. Decrease of the transmittance seems to be correlated with the cell sheet maturation. For the first time, we are reporting the success development of a device to estimate ASCCS harvesting time based on their transmittance.

Posology and Serum-/Xeno-Free Engineered Adipose Stromal Cells Cell Sheets

“…The ASCs kept growing over time, forming a strong multilayer cell sheet ( Figure 1A ). The transmittance of these undifferentiated ASCCSs decreased at around 60% as reported in a previous publication but using different culture media ( Ochiai et al, 2021 ). Osteoblast cell sheets were engineered in 15 days.…”

“…We understand that it is necessary to develop new tools and experiments to understand better the cell sheet physical properties as explained in this excellent review ( Efremov et al, 2021 ). Different physical properties could be studied, such as how the cell sheets can be stretched in one or two directions ( Homma et al, 2020 ; Huang et al, 2021 ), the nanoindentation ( Qian and Zhao, 2018 ), or optical properties ( Ochiai et al, 2021 ). Shimizu’s group reported that detached and stretched cardiomyocyte cell sheets from a temperature-responsive surface were longer, but that the cardiomyocyte cells had a unidirectional alignment.…”

“…The transmittance is measured only after the culture media are replaced, and it was measured only when the cells reached confluence until they form a multilayer cell sheet. The transmittance of the adipose stromal cells cell sheets was measured using the device described in the reference ( Ochiai et al, 2021 ). In summary, the cell culture dish is placed on the stage, always in the same position.…”

“…A light, produced by the light source, will shine through the cell sheet, and the light intensity will be measured by a detector placed under the stage. A single measurement is an average of 100 values per read from the detector with 10 reads per second, for 10 s per point (nine points per cell sheet) and per cell sheet ( Ochiai et al, 2021 ). The reference value was obtained by measuring the intensity of the light coming through a cell culture dish that has the same amount of culture media but with no cell sheet (blank sample).…”

“…In this study, we propose to engineer three different types of ASCCSs with chemically defined culture media (xeno- and serum-free culture media): undifferentiated, chondrocyte, and osteoblast multilayered cell sheets. In addition, using a transmittance device developed by our group, the difference in transmittance was studied to establish standard curves of the ASCCSs during their differentiation ( Ochiai et al, 2021 ). We also estimated the number of cells per cell sheet using the transmittance values, which is important information to provide to federal agencies in terms of posology.…”