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I need to establish a cell line starting from single cells seeded on a 96 wells plate by a FACS sorter. The cells I am using are human fibroblasts RPE-1 cultured in F12 medium supplemented with 10% FBS and antibiotics. After having seeded 5x96 cells (one cell per well in 5 plates) I have obtained only 4 colonies. Does anyone know any good (preferably personally tested) protocol to efficiently grow fibroblast colonies from single cells?

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  • $\begingroup$ Why do you sort the cells when you use a cell line? Additionally: What kind of colonies do you want? 2D, 3D? $\endgroup$ – Chris Jun 9 '15 at 10:05
  • $\begingroup$ I have engineered the cells using the CRISPR/Cas system and I sort the transfected one (GFP expressing vector). However, since only 1% of the transfected will have the right modification and since my final goal is to obtain a new cell line, I need to grow colonies out of single cells (2D, 3D doesn't matter), and then screen them to find the one carrying the correct modification. $\endgroup$ – alec_djinn Jun 9 '15 at 10:24
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This should be relatively straight forward. Sort the cells into single wells so all descendants are clones of the sorted cell and let them grow until they are dense enough to be transferred into a bigger plate (probably 48 or 24 well). Continue this process until you can go into big cell culture flasks or plates and make sure that you freeze away a number of aliquots as a backup if something goes wrong. Expand the cells further until you have enough cells to do an analysis.

Keratinocytes need some time to get highly proliferative (around a week or so) and if you don't grow them on a layer of feeder cells, usually also need a special conditioned medium. See the references below (if you have trouble accessing the papers, let me know).

What often also helps is the use of conditioned media. I know this from other cells in the skin which often don't like to grow (at least not as primary cells) unless you use conditioned media. For this, take of the media from the same sort of cells (here it would be keratinocytes) before it is completely used (typically yellow if the media contains pH indicators), centrifuge it to remove any cell debris and then mix it with fresh media to let your cells grow. You might need to experiment a bit with the mixing ratio between the "used" and the fresh media, but 50%:50% is usually a good starting point.

References:

  1. Isolation and cultivation of human keratinocytes from skin or plucked hair for the generation of induced pluripotent stem cells
  2. Culture techniques for human keratinocytes
  3. Improvement of human keratinocyte isolation and culture using thermolysin
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  • $\begingroup$ I did that but I got only 4 cells (out of almost 500) to grow into colonies and it took 3 weeks! Fibroblasts don't like to grow alone (at least this is what I am experiencing) so I was looking for suggestions about the protocol to use to make them grow well even if isolated.. I tried to seed different amount of cells but less than 1000 cells per well nothing grows. I will look to the links you posted. $\endgroup$ – alec_djinn Jun 9 '15 at 10:44
  • $\begingroup$ What media and what kind of plates do you use? $\endgroup$ – Chris Jun 9 '15 at 10:46
  • $\begingroup$ F12 medium supplemented with 10% FBS and antibiotics. Very standard 96 wells plates for cell cultures. The cells grow perfectly if I seed them in large number but a single cell will not grow. I think I need to tweak the medium, maybe use 50% conditioned medium... But before rushing in tens of tests I wanted to ask if someone have a nice running protocol to accomplish the task. $\endgroup$ – alec_djinn Jun 9 '15 at 10:50
  • $\begingroup$ That's what I would suggest as well, the use of conditioned medium. We rarely do keratinocytes, more melanocytes and melanoma and they often don't like to grow if there are not enough cells. $\endgroup$ – Chris Jun 9 '15 at 12:04
  • $\begingroup$ This is just to let all of you know that I am trying your suggestions. As soon I will have the results I will sign the correct answer. $\endgroup$ – alec_djinn Jun 19 '15 at 8:49
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Although I have not yet done, in general, cells are happier in a conditioning medium. Grow the original cells in 10cm dishes and save the medium and use it to grow single colonies. You might want to mix used and fresh medium (1:1 ratio).

Growth factors are another option. Insulin is sometimes used, but it also depends on cells.

Coating the surface of well with extracellular matrix such as collagen could be effective.

If your cells are normal human fibroblast, cells get senescent, stopping growing. You might want to use immortalized cells like hTERT RPE-1, although you should be careful because immortalization could cause characteristic changes.

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  • $\begingroup$ Thank you for the suggestion, I am indeed using hTERT RPE-1, I will try growing them in 1:1 conditioned medium and see.. $\endgroup$ – alec_djinn Jun 10 '15 at 7:30

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