Our OEM solutions for Flow Cytometry

Be in control of the full culture environment and its components, work with ease without the need for complex temperature control, and recover the cells for analyses effortlessly without harming them.

 

Why to choose our nanofibrillar cellulose and cellulase enzyme solutions as your OEM 3D cell culture solution?

1. No interference with downstream analysis

Our hydrogels contain only wood nanofibrillar cellulose fibers and ultra-pure water. In addition to the two main components of our hydrogels, the user can mix their components of choice (culture medium, proteins, growth factors etc.). This way the customer stays in full control of the cell culture environment and its components – see video, how our hydrogels can be used for setting up 3D cell culture assays. 

2. Recover your cells without harming them

Our cellulase enzyme degrades only nanocellulose fibers. The nanocellulose fibers turn into soluble glucose and the semi-solid hydrogel breaks down into a liquid solution. The cultured cells will be free from the 3D cell culture environment for downstream processing. The cellulase enzyme does not affect cell structures, cell viability or cell function during this process. 

3. Proven solution for automated liquid handling

There is no need for mechanical or temperature-based steps to break-down our hydrogels and recover your cells. Rather, simply use liquid handling to dispense our propriety cellulase enzyme solution and recover your cells.  

 

Cell structure retained

Images from Liisa Kanninen (Ph.D.) and Yan-Ru Lou (Ph.D.), University of Helsinki.

Images from Liisa Kanninen (Ph.D.) and Yan-Ru Lou (Ph.D.), University of Helsinki.

The three-dimensional structure is retained after recovering them from our nanocellulose hydrogels. Scanning electron microscopy (SEM) images of HepG2 spheroids revealing microvilli structure typical for hepatocytes. 

 

Cell function retained

Image adapted from Lou, Y.-R. et al., (2014).

Even the most sensitive cell types, like induced pluripotent stem cells and embryonic stem cells are not harmed by our enzymatic recovery. Both of the cells exhibited typical morphology and expressed the pluripotency markers OCT4 and SSEA-4.  

 

Cell viability retained 

Fibroblasts were cultured in 0.4% nanofibrillated cellulose (NFC) at 1x106cells/ml for 24hrs then cellulase enzyme degradation was performed. Cellulase enzyme was used at 300, 600 and 900μg/mg to degrade the NFC. The RealTime-Glo™ viability assay was added and the viability of the cells read through 20hrs of culture. 

 

How are 3D cell cultures analysed in downstream processing applications using flow cytometry? 

Flow cytometry allows for high-throughput analysis of large number of cells from 3D cell cultures. Fluorescent dyes are used in flow cytometry to quantify and identify cellular components and cell types. The analyzed cells are treated with one or more fluorochrome(s) and they undergo monochromatic excitations by one or more laser(s). The resulting fluorescence is collected by detectors in the machine and enables the analysis of cellular populations within 3D cell cultured spheroids.

Flow cytometry requires that the 3D cultured spheroids are disassembled into homogenous single-cell suspensions for analysis, which leads to loosing the spatial distribution of the marked cells in the spheroids. Recently there has been flow cytometry technologies that allow also whole spheroid analysis (e.g. Union Biometrica COPAS VISION).

Examples of flow cytometry applications are presented in Table 1 adapted from McKinnon, K.M. et al., (2018). 

Table 1: Examples of flow cytometry applications used in cell culture, adapted from [1]

Flow cytometry application Source of fluorescence
Quantifying cells in specific cell cycle phases Anti-Ki67, anti-PCNA, Hoechst 33342, 7AAD, and Chromomycin A3
Measure level of active proliferation Bromodeoxy uridine (BrdU) and conjugated anti-BrdU dye
Carboxyfluorescein succinimidyl ester (CFSE)
Quantifying cell viability Fluorescent diacetate (FDA), propidium iodide (PI), and Hoechst 33342
Identifying differentiation states and quantifying amount of differentiation Conjugated antibodies for differentiation markers
Quantifying cell types in cocultures Endogenous fluorescence in reporter cell lines
Identifying or isolating cell types in immunology Conjugated antibodies for lineage markers
Measuring cell activation and signaling Calcium indicators: Indo-1 and fluo-3
Measuring specific antigen response Biotinylated MHC multimers, in combination with fluorescent streptavidin
Identifying mechanism of cell death JC-1, anti-APO 2.7, fluorogenic caspase substrates, and anti-annexin-V
Measuring phagocytosis pH sensitive fluorescently tagged biomolecules or bacteria
Perform generational tracking Carboxyfluorescein succinimidyl ester (CFSE)
Measure level of protein expression Inducible expression of fluorescent protein

1. McKinnon, K.M. (2018). "Flow Cytometry: An Overview." Curr Protoc Immunol 120: p. 5.1.1-5.1.11.

Interested to OEM our wood nanocellulose based hydrogel or develop your own formulations?