Support

Culturing cells in 3D is generating a lot of interest and change from the traditional way of culturing cells in 2D is imminent. To make this transition as smooth as possible detailed information on the use of 3D cell culture solutions is required. Because of this, we have put together a collection of frequently asked questions, protocols used successfully with our products, publications where our products have been used as well as posters presented at various occasions, all available below.

Furthermore, we recomend you take a look at our "Recent Research" section for a collection of new and interesting articles on 3D cell culture using nanofiber scaffolds.

General Questions

Cells cultured in standard flat (2D) culturing flasks or wells tend to adapt to this unfamiliar environment by stretching out randomly along the surface and thus becoming more or less flat. This causes the cells to effectively lose roughly 50 % of their surface area in contact with surrounding culture medium as well as reducing possible cell-to-cell interactions by the same amount.

By creating a network of electrospun nanofibers for the cells to grow on, we provide the cells with a three dimensional (3D) environment, similar to collagen and elastin fibers of the extracellular matrix (ECM) that cells normally reside in.

Adding a third dimension to a cell’s environment creates significant differences in cellular characteristics and behavior such as differentiation, drug metabolism, gene expression and proliferation. Importantly, these differences normally create a greater similarity between the cultured cells and the living organism (e.g. human being) the cells are meant to represent – leading to more useful data and more relevant research.

For more information and a collection of relevant publications please have a look at www.3DCellculture.com.

Cellevate 3D scaffolds effectively mimics the structural properties of ECM and are easily tailored for specific applications through functionalization etc. Adaptable to work with just about any method of analysis applicable to standard 2D cell culturing alternatives, easing the impending transfer from 2D to 3D. Nanofiber scaffolds allows for a true 3D environment and allows cells to stretch and migrate throughout the whole scaffold material.

Cellevates scaffolds are produced with our patented high-throughput electrospinning technique which significantly improves well-to-well and batch-to-batch consistency and thereby reduce experimental variation. Our scaffolds provides cells with a true 3D micro-environment as opposed to many other 3D cell culturing products available on the market, that rather resemble more or less rugged 2D surfaces. Furthermore, Cellevates scaffolds are sterilized and ready to use out of the box and comes in a range of cell culturing formats according to industry standard sizes. Our products are compatible with light- and fluorescent microscopy and are designed to be compatible with both standard as well as high-throughput equipment.

Cellevate does provide custom scaffolds based on your experimentals needs, send us an e-mail at info@cellevate.com and we’ll discuss a specific solution that works for you!

Physical Properties & Formats

No. Cellevates scaffolds are single-use, disposable products.

No. However, for optimal results we recommend using the scaffolds within one year of purchase.

Certain chemicals may affect the fibers. Solvents such as acetone or toluene will deteriorate the fibers.

Cellevate offers a great variety of formats for cell culturing. All our scaffolds are available in both random- and aligned fiber orientation. Our single well dishes as well as 4-, 6- ,12- and 24-well culture plates consists of removable inserts that allows for easy imaging. Larger formats (48, 96 and 384) are designed with scaffolds fused to the multiwell-plate frame for easy handling and are suitable for larger screening experiments. Furthermore Cellevates 3D scaffolds are available mounted on crown inserts (without substrate backing) or as chamber slides. Alternatively, the scaffolds can be delivered as sheets in custom size or precut to fit your needs. All plates and dishes are of standard dimensions, and are compatible with standard laboratory plate readers and instruments.

Our standard scaffolds are made from synthetic, biocompatible poly-ε-caprolactone (PCL).

Yes. The engineers at Cellevate have experience working with a wide range of materials (synthetic and natural) such as: poly-lactic acid (PLA), polyvinyl-alcohol (PVA), polyamide-6 (nylon-6), polyvinylidene difluoride (PVDF), gelatin etc. Cellevate does provide custom scaffolds based on your experimentals needs. We can create custom scaffolds and tune characteristics such as material, fiber diameter, pore size and thickness to best suit your specific requirements. Do not hesitate to send us an e-mail at info@cellevate.com and we’ll discuss a specific solution that works for you!

Yes, PCL is biodegradable via hydrolysis, however in vitro the time for this is usually too long to be taken into account, and the structures can be treated as more or less inert.

The PCL fibers have an average diameter of 700 nm.

Cellevate produces nanofibers through a unique variation on electrospinning, allowing for precise fine-tuning of fiber parameters and excellent batch-to-batch consistency. Furthermore, all our products go through rigorous evaluation to assure the quality of each unit.

Usage

Cells representing all basic tissues except for muscle have been sucessfully cultured so far, e.g. Human neural precursor cells (HNPCs), retinal post-natal cells (RPNCs), L929 murine fibroblasts, human adipose tissue-derived stem cells (hASCs), circulating tumor cells (CTCs), BV-2 murine microglial cells, MCF10A human breast epithelial cell and human JIMT1 cancer cells etc.

We recommend the scaffolds to be wetted before use, this will allow the cells to properly migrate through the fiber network. Before adding your cells, we suggest soaking the fibers with sterile culture media, followed by incubation for at least 30 min, at 37°C. Please visit our support section for suggestions and protocols. The substrates can also be oxygen-plasma treated to further enhance the hydrophilicity and/or coated with adhesion promoting molecules such as fibronectin or laminin using standard protocols.

Yes, using standard procedures such as trypsin treatment etc, however, depending on cell type, cells may be captured inside the fiber scaffold, why several rinsing steps may be needed in order to retrieve all cells from the scaffolds. Please visit our support section for suggestions and protocols.

Yes. Please visit our support section for suggestions on how to use the scaffolds.

Yes, Cellevates scaffolds can be coated with most conventional methods. Please visit our support section for suggestions and protocols.

No maximum time limit has been found and the human neural precursor cells have been cultured successfully for many weeks.

Sterilization

Yes. All of Cellevates products are supplied sterile and ready to use.

No, the low glass transition temperature of poly-ε-caprolactone causes the fibers to deteriorate as a result of the heat and pressure of the autoclaving process.

Our collaborators have successfully used the following methods for sterilization: gamma irradiation, UV-sterilization and washing using a 70% ethanol solution.

Analysis & Imaging

Most standard methods of analysis are applicable. Standard imaging techniques, immunostainings, histological analysis, protein extraction and western blotting, cell viability assays, MTT, electrophysiological measurements using multiple electrode arrays (MEAs), absorbance and fluorescence based assays, etc. have all been performed successfully.

Optical microscopy (to some extent, imaging through the scaffold may be difficult), fluorescence microscopy, confocal microscopy, SEM, TEM, phase holographic microscopy.

No. No significant levels of autofluorescence have been measured for Cellevates scaffolds using standard excitation wavelengths. However, PCL exhibits a slight green autoflourescence during confocal microscopy. This is typically easy to exclude from images by adjusting threshold settings.

Scaffolds will give rise to a background signal when used in absorbance-based assays and fluorescence-based assays are thus preferred when possible. For absorbance based assays, well contents can be transferred and analysed separately.


Protocols


Protocol PDF
Cell lysis for protein extractionPDF
Cell recovery by trypsinationPDF
Cell viabilityPDF
Immunocytochemistry of cells in nanofiber scaffoldsPDF
Instructions for use of Cellevate 3D cell culture systemsPDF
Laminin coating of nanofiber scaffoldsPDF
Mounting nanofiber scaffolds for microscopyPDF
SEM fixation procedurePDF
Seeding cellsPDF
Western blotPDF

Publications


M. Castro-Zalis, S. Johansson, F. Johansson, U. Englund-Johansson, "Exploration of physical and chemical cues on retinal cell fate", Molecular and Cellular Neuroscience, Volume 75, September 2016, Pages 122–132

Maddaly Ravi, V. Paramesh, S.R. Kaviya, E. Anuradha, F.D. Paul Solomon, "3D Cell Culture Systems: Advantages and Applications", J. Cell. Physiol., Volume 230, Issue 1, January 2015, Pages 16–26

U. Englund-Johansson, E. Netanyah, F. Johansson, "Tailor-Made Electrospun Culture Scaffolds Control Human Neural Progenitor Cell Behavior — Studies on Cellular Migration and Phenotypic Differentiation", J. Biomaterials and Nanobiotechnology, Volume 8, Issue 1, January 2017, pages 1-21

A. Jakobsson, M. Ottosson, M. Castro-Zalis, D. O'Carroll, U. Englund-Johansson, F. Johansson, "Three-dimensional functional human neuronal networks in uncompressed low-density electrospun fiber scaffolds", J. Nanomedicine: nanotechnology, biology, and medicine, January 2017

Posters


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Controlling Retinal Cell Fate Using Nanotopography and Neurotropic Factors
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Into the Next Dimension – Exploration of 3D Nanofiber Scaffolds for Neural Stem Cells
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Nanofibers for Accelerated Wound Closure – a Regenerative Approach