by Dr. Susan Uprichard

Three-Dimensional Culture Model

​

Conventional adherent tissue culture involves growing cells on solid flat surfaces as two-dimensional (2-D) monolayers. Although such practices are routine and suitable for transformed or immortalized cell lines, de-differentiation and loss of specialized functions occurs when primary cells are removed from their host tissue and grown as 2-D monolayers. This is generally believed to be a result of the dissociation of primary cells from their native three-dimensional (3-D) structure in vivo to their 2-D propagation on flat impermeable substrates in vitro. As such, there is a continuing need to develop tissue culture systems which can either promote re-differentiation of laboratory cell lines or prevent primary cell lines from de-differentiating. The Rotary Cell Culture System (RCCS), a tissue culture bioreactor system manufactured by Synthecon, Incorporated (Houston, TX), addresses this need.

​

The Rotary Cell Culture System (RCCS)

​

The RCCS is a horizontally rotating cylindrical culture vessel, which offers an optimized suspension cell culture. Solid body rotation about a horizontal axis reduces the shear and turbulence generated by conventional stirred bioreactors minimizing mechanical cell damage and simulating aspects of microgravity, similar to the environment encountered during normal in vivo development. Thus, in contrast to conventional static tissue culture systems (e.g. flat culture flasks and dishes), cells grown in the RCCS are cultured in “suspended animation” where they are continuously free-falling, promoting the assembly of 3-D cellular aggregates, which allows for more efficient cell-to-cell interactions and exchange of growth factors. This in vivo-like environment promotes cell re-differentiation, allowing transformed and primary cell lines to become more structurally and functionally similar to their in vivo counterparts.

Our 3-D Cell Culture Application in the RCCS

​

One area where this is of particular relevance is the in vitro culturing of hepatocytes where research is limited by the unavailability of long-lived primary human hepatocytes and lack of highly differentiated hepatocyte cell lines. Relevant to our hepatitis C virus studies, we were interested in developing a more in vivo-like hepatocyte cell culture model to study this liver-tropic virus. Using the RCCS, we were able to culture Huh7 cells, an HCV-permissive human hepatoma cell line, three-dimensionally to promote cellular re-differentiation and polarization. Being able to assess HCV infection under these more in vivo-like conditions should prove useful for studying how HCV interacts with and alters hepatocyte function in a more physiologically relevant hepatocyte cell culture models.

Advantages of the RCCS

​

Importantly, Synthecon’s 3-D Rotary Cell Culture System is easy to use and a wide range of vessels are available, from small (1 mL) to large (500 mL), depending on your tissue culture needs. Alternatively, the Rotary Cell Culture Systems are available in multi-station 3-D bioreactors and custom designed products. While we initially chose to use commercially available collagen-coated microcarrier beads as a matrix for cell attachment, Synthecon also offers biodegradable polymer scaffolds, which are ideal for tissue engineering and might prove even more effective for culturing difficult primary cell lines, such as hepatocytes.

​

In summary, following extensive use, Synthecon’s 3-D Rotary Cell Culture System has proven to be an easy and efficient way of achieving more differentiated 3-D hepatocyte growth in vitro. Although the user should expect to invest a little more time when performing routine maintenance of 3-D cell cultures (e.g. media changes and sample collection), the results are superior to those achieved in more traditional 2-D cell culture.

​

Key Advantage

Synthecon’s RCCS provides anyone with the means of achieving more differentiated cell growth in vitro as it eliminates the negative effects of traditional static 2-D culture systems which often result in the loss of cell specific phenotypes and functions.

​

Main Challenge

Since culture conditions are cell-type specific and not universal, users may have to invest some time determining the most appropriate matrix and culture conditions for their specific cell line.

​

The Bottom Line

The RCCS is an easy to use and efficient cell culture system which promotes 3D cell growth, differentiation and cell-to-cell communication, and hence is ideal for anyone needing to culture their cells in a more in vivo like environment.