Hybridoma Re-cloning Introduction

Hybridoma re-cloning to obtain highly active positive hybridoma cells is an effective hybridoma rescue measure. Common methods for hybridoma re-cloning include limited dilution, semi-solid medium, soft agar, and automation system methods. Creative Biolabs has a well-established hybridoma platform that offers different approaches to hybridoma re-cloning.

Hybridoma Re-cloning Based on Limited Dilution Method

The limited dilution method is a simple and easy-to-perform technique. Textbooks and references generally recommend diluting cells to an average of 0.8 cells per well, when the probability of a well containing a single cell is highest. In practical studies, we recommend testing the optimal average number of cells per well for the experimental system used in conjunction with the support of cell growth by different experimental systems. The limited dilution method is performed as follows.

Positive cells are diluted to an average of a defined number per well. They are then seeded into 96-well plates containing feeder cells and incubated in a 5% CO₂ 37°C cell incubator. After about 5 days of observation, wells containing only single colonized cells are selected for supernatant assay. If the single-colony wells reach more than 50% and the selected single-colony wells test 100% positive, the cells are considered to be a monoclonal cell line. The cell line is named after the original well number of the hybridoma cells. The subcloned cell line is scaled up for ascites production. If the test result is not 100% positive, a second subcloning is required from the well with the highest positive value of the cell line obtained from this subcloning.

Hybridoma Re-cloning Based on Semi-solid Medium Method

The principle of semi-solid culture is somewhat similar to colony screening when doing molecular cloning. The specific steps of semi-solid culture are as follows: hybridoma cells are first mixed with liquid low melting point agarose or methylated cellulose solution and then poured onto a plate containing a pre-prepared feeder layer, and the medium is solid when cooled. After a period of incubation at 37°C, individual hybridoma cells form small clones resembling colonies. Cloning can also be achieved by using a pipette tip to pick out small clones onto culture plates to be cultured in a liquid medium.

Since the semi-solid medium can hold the cells in place during routine experimental manipulation, it does not affect the cells as they shift position. This is more conducive to accurately tracking the growth of individual cells into a clone. The disadvantage of this method is that it is more demanding and prone to cell non-growth or contamination.

Hybridoma Re-cloning Based on Automation System Method

With advances in technology, automated hybridoma screening and re-cloning techniques have emerged. Automated systems have greatly simplified the process and time for hybridoma screening and re-cloning, using automated technology to isolate and inoculate target cells. The method effectively avoids effective dilution and enables efficient and rapid picking of hybridomas and the establishment of stable cell lines.

Creative Biolabs is thrilled to share the key points and experiences in hybridoma re-cloning to facilitate the smooth running of your project.

All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.

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