Types of Subculture of Cell: 2 Types (With Diagram)

This article throws light upon the two types of subculture of cell. The two types of subculture of cell are: (1) Monolayer Cultures and (2) Suspension Cultures.

Subculture (or passage) refers to the transfer of cells from one culture vessel to another culture vessel. Subculture usually (not always) involves the subdivision of proliferating cells that enables the propagation of a cell line.

The term passage number is used to indicate the number of times a culture has been sub-cultured.

The standard growth curve of cells in a culture is depicted in Fig. 36.5. The initial lag phase is followed by exponential or log phase and a plateau phase. During the active growth period in the log phase, the medium must be changed frequently, or else growth ceases. As the cell concentration exceeds the capacity of the medium, the culture has to be divided to subculture.

There are two types of subcultures-monolayer subculture and suspension subculture.

Type # 1. Monolayer Cultures:

When the bottom of the culture vessel is covered with a continuous layer of cells, usually one cell in thickness, they are referred to as monolayer cultures. The attachment of cells among themselves and to the substrate (i.e. culture vessel) is mediated through surface glycoproteins (cell adhesion molecules) and calcium ions (Ca²⁺). For sub-culturing of monolayer cultures, it is usually necessary to remove the medium and dissociate the cells in the monolayer by degrading the cell adhesion molecules, besides removing Ca²⁺.

Methods of Cell Dissociation:

There are physical and enzymatic methods for dissociation of monolayer cultures (Table. 36.3).

Mechanical shaking and cell scraping are employed for cultures which are loosely adhered, and the use of proteases has to be avoided. Among the enzymes, trypsin is the most frequently used. For certain cell monolayers, which cannot be dissociated by trypsin, other enzymes such as pronase, dispase and collagenase are used. Prior to cell dissociation by enzymes, the monolayers are usually subjected to pretreatment of EDTA for the removal of Ca²⁺.

Criteria for Subculture of Monolayers:

The sub-culturing is ideally carried out between the middle of the log phase and the time before they enter plateau phase (Fig. 36.6). Subculture of cells should not be done when they are in lag phase. The other important criteria for subculture of monolayers are briefly described.

Culture density:

It is advisable to subculture the normal or transformed monolayer cultures, as soon as they reach confluence. Confluence denotes the culture stage wherein all the available growth area is utilized and the cells make close contact with each other.

Medium exhaustion:

A drop in pH is usually accompanied by an increase in culture cell density. Thus, when the pH falls, the medium must be changed, followed by subculture.

Scheduled timings of subculture:

It is now possible to have specified schedule timings for subculture of each cell line. For a majority of cell cultures, the medium change is usually done after 3-4 days, and sub-culturing after 7 days.

Purpose of subculture:

The purpose for which the cells are required is another important criteria for consideration of sub-culturing. Generally, if the cells are to be used for any specialized purpose, they have to be sub-cultured more frequently.

Techniques of Monolayer Subculture:

The subculture of monolayer cells basically consists of the following steps (Fig. 36.6)

1. Removal of the medium

2. Brief exposure of the cells to trypsin.

3. Removal of trypsin and dispersion in a medium.

4. Incubation of cells to round up.

5. Re-suspension of the cells in a medium for counting and reseeding.

6. Cells reseeded and grown to monolayers.

Cell concentration at subculture:

Most of the continuous cell lines are sub-cultured at a seeding concentration between 1 × 10⁴ and 5 × 10⁴ cells/ ml. However for a new culture, subculture has to be started at a high concentration and gradually reduced.

Type # 2. Suspension Cultures:

Majority of continuous cell lines grow as monolayers. Some of the cells which are non- adhesive e. g. cells of leukemia or certain cells which can be mechanically kept in suspension can be propagated in suspension. The transformed cells are sub-cultured by this method. Subculture by suspension is comparable to culturing of bacteria or yeast.

Advantages of Cell Propagation by Suspension:

i. The process of propagation is faster.

ii. The lag period is usually shorter.

iii. Results in homogeneous suspension of cells.

iv. Treatment with trypsin is not required.

v. Scale-up is convenient.

vi. No need for frequent replacement of the medium.

vii. Maintenance is easy.

viii. Bulk production of cells can be conveniently achieved.

Criteria for Suspension Subculture:

The criteria adopted for suspension subculture are the same as that already described for monolayer subcultures.

The following aspects have to be considered:

i. Culture density.

ii. pH change representing medium exhaustion.

iii. Schedule timings of subculture.

iv. Purpose of subculture.

Technique of Suspension Culture:

The cells can be suspended in a culture flask (a stirrer flask) containing the desired medium (Fig. 36.7). The medium is continuously stirred with a magnetic pendulum rotating at the base of the flask. The cells have to periodically examined for contamination or signs of deterioration.