The value of cell culture experiments lies in how well they represent the native microenvironments of in vivo systems. Among factors that significantly influence the cellular behavior of in vitro models, the surface properties of the solid culture support are critical. While adequate for some applications, conventional plastic culture surfaces pose limitations in studies of more complex physiological processes, such as cell migration, cell-cell communication, and cell polarization. This has led to the development of membrane-based cultureware that more closely recapitulate in vivo conditions.
Download Podcast
In this podcast, Jun Park, Ph.D., R&D Manager, Cell Biology Group at MilliporeSigma, talks about membrane-based culture systems and the applications they support. In the process, he clarifies some misconceptions and provides some useful tips and tricks.
Microporous membranes have been used since the early 1950s to create in vitro systems that better mimic in vivo phenomena. In 1962, the Boyden chamber, named after the study’s author, used a microporous membrane designed and manufactured by the Millipore® Corporation to measure the chemotactic effect of tuberculin on rabbit granulocytes in a two-chamber system. Since then, Millipore® engineers have introduced a range of membrane-based culture solutions, with Millicell® inserts as a diverse, highly recognized brand in advanced in vitro systems.
Unlike 2D plastic surfaces, microporous membranes offer many advantages that support a more physiological culture environment. These systems utilize a permeable membrane to create two separate chambers, allowing cells to experience different conditions. Adherent cells can be cultured on either the top (apical) or the bottom (basolateral) side of the membrane—or co-cultured in more complex studies involving multiple cell phenotypes that may incorporate suspension cells. Compartmentalization allows cells to grow in a polarized fashion and form tight junctions, as seen in an epithelial monolayer. The two chambers are also ideal for creating chemical gradients for studies of cell migration and invasion across the membrane (Figure 1).
Figure 1. Illustration of how porous membrane system can be used to model monolayer formation and cell migration. Numbers indicate recommended pore size in microns.
The Millicell® cell culture product family includes 24-well and 96-well insert plates, as well as hanging and standing single-well inserts. The choice of membrane insert is application-dependent, and takes into consideration pore size, membrane fabrication chemistry, pore density, and optical clarity.
Pore size: Membrane pore size ranges from 0.4 µm to 12 µm, and the right choice varies depending on the cell type and assay. As a general guide, membranes with pore sizes greater than 3 µm are recommended for migration, invasion, and chemotaxis assays, while smaller pore sizes are best suited for modeling epithelial polarization in tissue models.
Membranes: Millicell® inserts are available in four different chemistries: polytetrafluoroethylene (PTFE), mixed cellulose ester (MCE), polycarbonate (PC), and polyethylene terephthalate (PET) (Figure 2). Unlike other membrane types, PTFE is a low protein-binding membrane and must be pre-coated with extracellular matrix (ECM) protein prior to seeding cells for culture. While it’s not required, MCE, PC, and PET membranes may benefit from ECM coating for some assays. Both PTFE and MCE have small pore sizes (0.4 µm), making them ideal for epithelial barrier studies.
PC and PET membranes are both manufactured using a process of track etching, which allows for a variety of pore sizes (0.4–12 µm and 0.4–10 μm for PET) and pore densities (105–108 pores/cm2). As a result, PC and PET membranes are amenable to a broader range of assays, including those that require larger pore sizes (e.g., migration/invasion assays). PC and PET are also significantly thinner than other membranes (10–20 µm), which is optimal for creating more physiologically relevant assays.
Figure 2. Insert membrane options and associated features.
Pore density & optical clarity: PC and PET membranes come in a range of pore sizes and densities. This means a membrane of a given pore size can have a low (105 pores/cm2) or a high (108 pores/cm2) pore density. Cells prefer higher pore densities for growth on solid surfaces. However, pore density is inversely proportional to optical clarity due to light scattering, and can therefore interfere with imaging. The optimal balance between cell growth and optical clarity depends on the assay, and on whether imaging is required.
Microporous membrane systems enable groundbreaking experiments with organotypic models. For this application, Millicell®-CM inserts offer an optically clear, PTFE membrane with a proprietary coating to enhance cell adhesion.
Millicell®-CM inserts have been used in a range of published studies to model epithelial barriers and, more recently, for the culture of organoids. In a 2017 study published in the journal Nature Methods, Seet et al. differentiated hematopoietic stem cells to mature T cells using an artificial organoid system on a Millicell®-CM insert.
Figure 3. Skin differentiation at the air-liquid interface of Millicell® insert.
In a more recent study published in 2019, Giandomenico et al. cultured cerebral organoids at the air-liquid interface of a Millicell® insert, with improved physiological and functional results. The air-liquid technique can also be used to differentiate and grow organotypic skin models on the Millicell® system (Figure 3). Using a 14-day protocol, Millipore® scientists grew a healthy epidermis with 10 layers, which can mimic in vivo skin models for absorption and toxicity testing.
Membrane-based culture systems foster a more in vivo-like cell growth environment and support a range of applications including primary and secondary screening, transport assays, toxicity screening, cell signaling, cell proliferation, and ADME/toxicity drug studies.
Booth R, Park J, Chen C, Clark R. Cultural evolution: Towards more predictive in vitro models of biological systems. MilliporeSigma. 2020
Boyden S. The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes. J Exp Med. 1962;115(3):453–466. doi:10.1084/jem.115.3.453
Giandomenico SL, Mierau SB, Gibbons GM, et al. Cerebral organoids at the air-liquid interface generate diverse nerve tracts with functional output. Nat Neurosci. 2019;22(4):669–679. doi:10.1038/s41593-019-0350-2
Seet CS, He C, Bethune MT, et al. Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids. Nat Methods. 2017;14(5):521–530. doi:10.1038/nmeth.4237