Question

Arrange the steps involved in the maintenance of embryonic stem (ES) cell culture in the correct sequence:
A. Prepare feeder plates two days in advance
B. Passaging is done at 1:4 ratio
C. Undifferentiated ES cells require 37° C, 5% CO2, RH 85-100% since they can differentiate spontaneously
D. Trypsinisation or mechanical cutting is done
E. Start feeding 48 hrs after thawing/passaging, every day
Choose the correct answer from the options given below:

• A. A, C, D, E, B
• B. C, A, B, E, D
• C. C, A, D, B, E
• D. B, D, C, E, A

Hint:

ESC Culture Workflow: Feeder plate preparation (A) $\rightarrow$ Incubate under strict $5\%\text{ CO}_2$ (C) $\rightarrow$ Trypsinize/dissociate colonies (D) $\rightarrow$ Daily feeding (E) $\rightarrow$ Split passaging (B).

Answer 1

The Correct Option is A

Step 1: Understanding the Concept:
Embryonic stem cell (ESC) maintenance protocols ensure continuous pluripotency and prevent spontaneous lineage differentiation.

Step 2: Detailed Explanation:
Standard laboratory operating protocol for maintaining mouse/human embryonic stem cell cultures:
1. Step 1 (A): Prepare primary mouse embryonic fibroblast (MEF) feeder layer plates coated with gelatin 2 days in advance (mitotically inactivated with mitomycin C).
2. Step 2 (C): Maintain undifferentiated ESC colonies in a humidified incubator at $37^\circ\text{C}$, $5\%\text{ CO}_2$, and $85-100\%\text{ RH}$ with LIF/bFGF pluripotency supplements.
3. Step 3 (D): When colonies reach $70-80\%$ confluence, perform gentle trypsinization or mechanical cutting/dissociation into small cell clumps.
4. Step 4 (E): Initiate daily media changes starting 48 hours post-thawing/plating.
5. Step 5 (B): Subculture and routine passaging at a split ratio of $1:4$.
Sequence: A $\rightarrow$ C $\rightarrow$ D $\rightarrow$ E $\rightarrow$ B.

Step 3: Final Answer:
Therefore, the correct sequence is A, C, D, E, B, matching option (A).