Labware

Key issues to be aware of when collecting cell-sheet.

1. During cell culture

It is imperative that cells you are trying to collect as "cell-sheet" is covering entire surface of UpCell (over-confluent and cell-cell junction is firmly established) before you attempt to detach cells as a "cell-sheet". Some cells exhibit easier detachment property when cultured until beyond stratification. When there is only loose connection between cells, it may be difficult to obtain strong cell-sheet. It is highly recommended that cells are cultured beyond over-confluency.

2. Detachment

Time
Beware of difference in time required to detach cells by its type. Some cells come off in 10-15 min (such as NIH/3T3) whereas others require 30 min (such as cornea epithelium cells).

Temperature
Please pay extra attention to the temperature for cell detachment. The hydrophilicity of the surface of UpCell is constant below 20-25℃, therefore please avoid leaving UpCell at extreme low temperature such as 4℃. At extreme low temperature, cells lose its ATP metabolism and make them difficult to detach. ATP metabolism is essential when detaching cells since cell detachment from UpCell surface is a function of "surface becomes hydrophilic" thus "cells lose their anchorage" and "get rounded and lift off from surface using its metabolism".

3. Transfer of cell-sheet

When transferring detached cell-sheet to host using the membrane, it is important that adherence property of "cell-sheet and the membrane" is weaker than that of "cell-sheet and the host". It follows that method or the time required to transfter cell-sheet to the host varies depending on the type of host. The time and method is less complex when transferring cell-sheet to rather sticky animal organs than to the bare cell cultureware.
Longer time or some kind of weighing may be necessary to make cell-sheet firmly attach to the the new surface. Additional caution is required on following points.

[a] Make sure there is no air bubble in between cell-sheet and the membrane. Tight adherence between cell-sheet and the membrane influence the success of cell-sheet detachment from the UpCell surface.

[b] Be careful of the amount of media to be added when detaching cell-sheet from UpCell surface using membrane to prevent cells from drying. Recommended amount of media is 50uL for UpCell 3.5 cm surface.

[c] Add 1mL of culture media on the membrane after cell-sheet transfer to new host. This will weaken the hygroscopicity of the membrane to release cell-sheet off the membrane. Make sure cell-sheet is firmly attached to the surface of the host before removal of the membrane. Depending on the type of the surface of the host, longer placement time or weighing may be required. Transferring technique can dramatically be improved by practicing the proceudre using such sticky gel like acrylamide as the host.