To verify the pluripotency of established iPS cells, teratoma formation assays are performed by injecting these iPS cells into the testis or intramuscular in immunodeficient mice. Formed teratomas are observed whether differentiation into the three germ layers from iPS cells has achieved or not. This repository stores and shares histological images generated from these teratoma formation assays, with the aim of contributing to the development of AI systems capable of automatically assessing pluripotency.
Fixation Procedure
1. Dispense 30 mL of 4% PFA into 50 mL tubes to fix the excised teratomas. Use one tube per mouse.
2. Cover the workbench with Kimwipes and prepare dissection tools.
3. Remove the mouse from the cage and perform cervical dislocation.
4. Thoroughly moisten the entire body, especially the abdominal area, with ethanol to prevent fur from scattering.
5. Grasp the skin with forceps and use scissors with rounded tips to make large vertical and horizontal incisions from the abdomen toward the legs to expose the internal tissues.
6. Excise the teratomas from the testes or legs, trimming off excess fat and any surrounding membranes containing fluid. If the teratoma is too large to fit into the 50 mL tube, cut it in half.
7. Rinse the teratoma briefly with PBS and place it into a 50 mL tube containing 4% PFA.
(Normally, all teratomas from one mouse are placed in a single tube. If there are too many, they may be divided into two tubes.)
8. Store the teratomas in 4% PFA at 4ºC in a designated toxic/controlled substance refrigerator for 1–2 days to fix them.
9. Prepare new 50 mL tubes containing approximately 30 mL of 70% ethanol. (Since this is for HE staining, Milli-Q water can be used to make the 70% ethanol.)
10. Using forceps, gently transfer the teratomas from the 4% PFA to the 70% ethanol, being careful not to damage them and to minimize carryover of PFA.
11. Store the teratomas in 70% ethanol at 4ºC or at −30ºC for long-term storage.
12. For teratoma analysis, outsource the embedding, sectioning, and HE staining procedures.
Scanning procedure
Scanning Device: NanoZoomer210
Objective Lens: 20× N.A. 0.75 (equivalent to 40× mode)
Sample Size: 26 mm × 76 mm
Sample Thickness: 0.9 mm to 1.2 mm
Scan Resolution: 0.23 μm/pixel
Focusing Method: Pre-focus map
Z-stack Function: Available
Image Compression Format: JPEG compression, uncompressed image (8 bit)
Virtual Slide Format: JPEG-compressed image + slide information