Historically, cell line development has predominantly relied on a limited number of cell lines such as CHO-K1, HEK293T, and HeLa. However, as biotechnology continues to advance, there is a growing demand for cell line development platforms that can support a broad range of cell types.
To demonstrate the power and versatility of CellRaft Technology to enable clonal cell growth, our team set an ambitious goal to test and characterize 100 unique cell lines on the CellRaft Array. We successfully accomplished this milestone when we added the 100th cell line in December 2022.
During this endeavor, our team gained valuable insights into the specific requirements of each individual cell line. Here are four key points we learned along the way:
1. Flask-like culture conditions enable the culture and cloning of lines that are not amenable to traditional methods.
Some cell lines are very difficult to clone using traditional cloning methods that place a single cell alone in a well, creating a lack of important growth factors and mitogens from neighboring cells. The CellRaft Array provides a unique cloning environment where single cells can divide in a flask-like bulk culture environment and share contiguous media while maintaining spatial segregation for monoclonality. Single cell cloning efficiency was greater than 70% for most of the cell lines that we tested, compared to the typical 0-30% cloning efficiency of a traditional limiting dilution workflow (Figure 1). When tested against limiting dilution, the CellRaft Array yielded hundreds more confirmed clones (Figure 2).
Figure 1: Single cell cloning efficiency of various cell types tested on the CellRaft Array. Defined as the percentage of single cells that formed clones, single cell cloning efficiency was greater than 70% for most cells tested.
Figure 2: Total number of single cell derived clones obtained from various cell types on one CellRaft Array or three limiting dilution plates per cell line. The CellRaft Array outperformed limiting dilution by generating hundreds more single cell derived clones. MHH-ES-1 cells failed to proliferate in limiting dilution plates but yielded 867 single cell derived clones on just one CellRaft Array.
2. We were able to generate more clones while significantly reducing time, media, and consumables.
One CellRaft Array can yield thousands of clones in a workflow that, for most cells, requires less than three weeks from seeding cells on the CellRaft Array to a full 96-well plate of expanding clones. Renca cells seeded on a 100 μm Quad-Reservoir CellRaft Array yielded 2,710 single cell derived clones after only three days of incubation.
Following the isolation of CellRafts containing monoclonal colonies to a 96-well plate, cells began to expand off-raft after only three days.
Assuming a generous limiting dilution efficiency of 15 clones per 96-well plate, a limiting dilution workflow to obtain a similar number of clones would require 181 96-well plates, over 1700 mL of media, and countless hours of hands-on time. If Renca cells required an extracellular matrix-like Matrigel, only 2 mL would be required to coat the CellRaft Array compared to 869 mL required to coat 181 96-well plates.
3. Each cell line requires characterization for optimal results.
Cells vary widely in morphology, preferred seeding density, and extracellular matrix requirements. While some cells like iPSCs prefer the extra growth area of the 200 μm CellRafts, other cells prefer the higher seeding density that the 100 μm CellRafts offer. Cells also range in doubling time, from less than 10 hours for BHK-21 cells to more than 40 hours for PC-12 adrenal cells, requiring modulation of imaging frequency and time on the array until isolation into a collection plate (Figure 3).
Figure 3: BHK-21 cells proliferating on a 200 μm CellRaft and PC-12 cells proliferating on a 100 μm CellRaft. BHK-21 cells prefer the larger 200 μm CellRaft and require only two days on the CellRaft Array before isolation to a 96-well collection plate. Slow-growing PC-12 cells prefer the smaller 100 μm CellRaft and are allowed to proliferate for at least six days before isolation.
4. The requirements of a cell line in traditional flask culture may differ compared to its needs on the CellRaft Array.
When initiating the culture of any cell type on an extracellular matrix, various matrix concentrations should be tested to determine an optimal concentration. Various concentrations of Matrigel, a common extracellular matrix used to coat tissue culture surfaces, were tested on the CellRaft Array with a stable iPSC line. Single cell cloning efficiency increased from 49% to 74% with increasing concentrations of Matrigel (Figure 4). While cell lines typically prefer a higher extracellular matrix concentration when propagated clonally on the CellRaft Array, only 2 mL of dilute matrix is required for coating while at least 14 mL of matrix is required to coat three limiting dilution plates.
Figure 4: Single cell cloning efficiency of iPSCs on the CellRaft Array with increasing concentrations of Growth Factor Reduced Matrigel (Corning).
Lessons Learned
In our endeavor to test 100 different cell lines using CellRaft technology, we discovered that the success of each cell type relies on meticulously characterized conditions. Variations between cells influence the optimal CellRaft Array size, seeding density, and extracellular matrix required to get the most out of cloning cells. One prevailing theme that emerged from our testing of 100 cell lines was that cells are much easier to clone when they can share media with neighboring cells, and happy cells make happy scientists.
Go to our Cell Atlas to see all 100 cell lines that we tested, and let us know if we haven’t tested yours yet!
Anna Lane, B.S.
Anna works on the Product Applications team where she develops novel single-cell workflows that are compatible with the CellRaft AIR system. Before joining Cell Microsystems, she worked as an undergraduate research assistant in a microbiology laboratory and as an intern on a quality control team at Lonza Biologics. She holds a Bachelor of Science in Microbiology and Biochemistry from the University of Maine.
