Adherent cells have extracellular matrix comprising of adhesion proteins that anchor them to the vessel they are in. To sub-culture them, they need to be detached either mechanically or enzymatically, with trypsin enzyme acting as a proteinase by breaking the adhering proteins. They are primarily used for cytology.

Suspension cells, on the other hand, remain free-floating in the medium without requiring any enzymatic or mechanical action. They are circular in structure and commonly used for bulk protein production and batch harvesting.

The Significance of Subculturing:

Sub-culturing occurs through passaging, where cells from a previous culture (a primary culture) are shifted into fresh growth medium. This is necessary when the space in the culture flask reaches 50-80% confluency, indicating 50-80% coverage of the medium. This transfer allows for the expansion of the population, especially for adherent cells, where the available surface area limits their growth, and for suspension cells, where the concentration of cells in the medium limits their growth.

K-562 Cells:

K-562 are cell lines originally extracted from a 53-year-old patient with chronic myeloid leukemia. These ligands range from 15-20 μm in diameter and appear round when viewed using scanning electron microscopy. They are suspension cells and float freely in media. Although their nature was an issue of debate in 1975, further research was able to conclude that K562 cells are multipotential hematopoietic cells that can differentiate into progenitors of erythrocytes, granulocytes, and monocytes.

HaCaT Cells:

HaCaT cells are spontaneous immortalized human keratinocyte cells that adhere to the surface of the vessel they are in through the extracellular matrix they secrete.

The Aseptic Technique:

Materials used for the process of sub-culturing adherent and suspension cells include T-25 and T-75 flasks.

Preparation of the Laminar Flow Hood includes 20-25 minutes of the UV light being switched on for thorough sterilization. The cabinet is initiated by switching on the light and the fan, which pulls in air and filters it through a HEPA filter for airborne contamination elimination. All materials used inside the hood are sterilized with 70% ethanol. The gloves are sterilized above and below.

Sub-culturing of suspension cells (using K-562 cells) and adherent cells (using HaCat cells) requires detachment with the requisite procedure.

In the T-75 flask, the leftover contents were treated with 2mL of trypsin, followed by incubation at 37°C for 5-10 minutes. Trypsin is known to break down the proteins constituting the extracellular matrix and therefore caused the cells to detach from the flask. After completion of the incubation, gentle tapping of the flask from the side, with the neck facing upwards, was done to ensure all cells were dislodged from the flask. Adding 10mL of media, which contains proteases, to the flask helped in deactivating the trypsin.

Centrifugation was then performed at 1500rpm for 5 minutes to separate the liquid contents from the cells, while keeping the cells alive. The cell pellet was subsequently suspended using 10mL of media by aspirating the liquid and drawing it up and down two to three times until the pellet was broken. The solution was pipetted into the desired flasks after resuspending the cells.

Several precautions were observed during the process, including not incubating trypsin for more than 10 minutes when sub-culturing adherent cells to prevent damage to proteins, venting the cells while storing by loosening the cap and ensuring each flask was facing upwards.

Contamination was apparent in the cultures in the form of tiny specs in the micrograph, as labeled.

Sustained exposure to trypsin can cause cells to transition to a state of reversible stemness amenable to transdifferentiation. Several research papers such as Maryada Sharma et al. (2019), Klein et al. (1976), Lozzio et al. (1981), and Lozzio et al. (1975) are essential in developing cell biology methods.