Microcavity-assisted cloning (MAC) of hard-to-clone HepG2 cell lines: cloning made easy

Abstract

Cloning is a key molecular biology procedure for obtaining a genetically homogenous population of organisms or cell lines. It requires the expansion of new cell populations starting from single genetically modified cells. Despite the technical progress, cloning of many cell lines remains difficult. Cloning often fails either due to the strenuous conditions associated with manipulating cells or because many cells don't tolerate a single-cell state. Here we describe a new cloning method utilizing low adhesion microcavity plates. This new technique, named microcavity-assisted cloning (MAC) was developed to clone difficult-to-clone HepG2 cells. The clones were produced following CRISPR/Cas9 knockout of the GSTA1 gene by a random distribution of 200, 400, and 800 cells into 550 microcavities of a 24-well low adhesion plate originally designed for the culture of spheroids. The knockout of GSTA1 was verified at the protein level using Western blotting. The advantages of the MAC method are its low cost, ease of the procedure, and the possibility of scaling up the throughput and automatization.

Keywords: Cell line; Cloning; Hard-to-clone; HepG2; Microcavity; Single cell; Sorting.

Fig. 1

Workflow of the MAC cloning procedure. A Cells are resuspended. This step is critical, particularly in cell cultures that tend to aggregate. B 100 to 200 cells are deposited in each well containing 550 low-adhesion cavities. C Cells are incubated at 37⁰C for 10 days until the spheroids form. D Clone recovery. Using a standard laboratory pipet, spheroids can be transferred to a standard cell culture plate with the adhesive surface for outgrowth and testing

Fig. 2

Distribution of cells in the microcavities of Elplasia 24-well microcavity plate after deposition of 100 (A), 200 (B), 400 (C), and 800 (D) cells per well. The expected distributions are shown in black and the observed distributions are in grey. The ideal ratio of one to multiple cells per well was obtained when depositing less than 200 cells per well. The error bars correspond to the mean plus or minus one standard deviation

Fig. 3

A Spheroid growth was monitored for 20 clones by measuring the clone surface area on indicated days. B Images show the growth of a spheroid through time

Fig. 4

Proof-of-concept of the MAC method for CRISPR/Cas9 ko mutants. Western blot to detect GSTA1 in 19 clones obtained with MAC method. Out of 19 clones screened, 11 were GSTA1 knockouts confirming the ability of the method to isolate clonal cell populations. L = ladder (25 kDa)