article : Cloning Process

Cloning - Cells derived from a single cell through mitosis constitute a clone and the process of obtaining clones is called cloning. (Asexual progeny of a single individual make up a clone).

In simple terms, cloning consists of trypsinisation of a monolayer culture to prepare a cell suspension, 3-4 dilution steps to achieve a suitable cell density (10-200 cells/ml), and seeding in Petri dishes or flasks or multi well dishes. The culture vessels are incubated for 1-3 weeks with a medium change after 1 week; by this time colonies will develop.

Colonies may be isolated

(1) directly from multi well dishes by trypsinisation of usually such wells, which contained only one cell at the start, or which have a single distinct colony originating from a single cell lying away from other nondividing cell or cells. This is confirmed by microscopic observation.

(2) Isolation of clones from petriplates is done by using cloning rings, which are placed around the desired colonies after the medium is poured. The cell colony within each porcelain, teflon or stainless steel ring is trypisnized, cells are suspended in medium and seeded in a well of multiwell plate or in a flask. Alternatively,

(3) the desired colony may be shielded and the remaining colonies are irradiated by a lethal dose (3,000 rads) of X-rays or gamma-rays. The protected colony is trypsinised, and the cells are cloned in the same plate; the irradiated cells serve as a feeder layer.

Plating efficiency (per cent of cells forming colonies) of continuous cell lines is generally 10% or higher, while that of finite cell lines may be quite low, say, 0.5-5% or even zero.

Several approaches have been used to enhance the plating efficiency, e.g.,

(i) use of a rich medium,

(ii) use of serum, especially foetal calf serum, in the medium,

(iii) using conditioned medium (a medium in which homologous cells were grown for a period of time),

(iv) use of feeder layers,

(v) addition of hormones like insulin, dexamethasone, etc.,

(vi) providing intermediate metabolites like keto acids, nucleosides, etc.

A feeder layer is a layer of cells, which have been treated to prevent their growth and ultimately cause their death (cells irradiated with X-rays or gamma-rays at 3,000 rads die in 3 weeks); these cells, however, provide the necessary metabolites to enhance the plating efficiency of cells being cloned.

A feeder layer may be established by trypsinising embryo fibroblasts from a primary culture and reseeding the cells at 10⁵ cells/ml density. When the monolayer reaches 50% confluence (50% of the substrate surface is covered by the monolayer) the cells are either treated with mitomycin-C (0.25 μg/ml solution at the rate of 2 μg/10⁶ cells) overnight, or are irradiated with 3,000 rad X-'rays.

After treatment, the cells are incubated for 24 hours in fresh medium, tryspinised and reseeded at 5 x 10⁴cells/ml, and incubated for 24-48 hours. This yields the feeder layer on which cells to be cloned are seeded, incubated and desired colonies isolate.

Feeder layer may be established using homologous cells (cells from the same species), but it is perferable to use heterologous cells for easy detection of accidental cross contamination in the isolated clones.

Cloning is used to

(i) obtain homogeneous cell lines from heterogeneous cell cultures,

(ii) to isolate biochemical mutants and

(iii) cell strains with marker chromosomes, and

(iv) to develop hybridoma clones.

Cloning is generally applied to continuous cell lines, but often their clones become considerably heterogeneous by the time they are sufficiently multiplied for use. The problem with finite cell lines is that of life span; by the time the clone is sufficiently multiplied, the cells may be approaching sensescene.

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