The Science of Alvetex

Cell-to-cell Interactions and Organization

Cells grow and divide occupying the 3D space within the porous Alvetex scaffold. Cells form complex interactions with one another, behaving in a manner that far more closely mimics normal growth in tissues than is possible using traditional 2D techniques.

Cells are free to migrate throughout the scaffold, functioning as they would within their natural environment, laying down extracellular matrix and forming organized and complex in vivo-like structures. This often leads to the formation of “mini slabs” of tissue.

Alvetex derived cell cultures can be processed just like normal tissue samples and prepared for histology using standard procedures including fixation, embedding, thin sectioning and counter-staining.

Above: 3D growth of cultured cells form 200 μm thick ‘slabs of tissue’. A: Naked Alvetex Scaffold before cell seeding viewed under scanning electron microscope to show the highly porous scaffold. B: Cells have grown throughout Alvetex Scaffold to the point where the scaffold is no longer visible. C: Hepatocytes form a thick multilayer on top of Alvetex Strata with maximal cell-cell contact as would be found within in in vivo tissue.

Above: Tissue processing and staining highlight the complex organization of cells growing through-out Alvetex Scaffold. D: Resin sections of Alvetex Scaffold showing skin keratinocytes stained with Toluidine Blue. E: Paraffin sectioned skin keratinocytes counter stained with H&E viewed by bright field microscopy.

Improved Cell Function

Alvetex 3D cell culture enables cells to maintain their natural morphology and and form complex organization, leading to improved cell function and responsiveness which is much more representative of the natural in vivo environment.

Alvetex delivers data of unmatched biological relevance, and provides a much deeper understanding of how cells function in vivo. Factors such as cell viability and responsiveness have been demonstrated to be enhanced when growing cells in Alvetex in comparison to 2D monolayer cultures.

* Data generated during a collaborative project between Reinnervate Ltd and LGC Standards – data now published in the following journal: Title: Rat primary hepatocytes show enhanced performance and sensitivity to acetaminophen during three dimensional culture on a novel polystyrene scaffold designed for routine use. Maaike Schutte, Bridget Fox, Marc Baradez, Alison Devonshire, Jesus Minguez, Maria Bokhari, Stefan Przyborski, Damian Marshall. Assay and Drug Development Technologies. DOI: 10.1089/adt.2011.0371

Examples of Cell Types Cultured on Alvetex

Following is an inexhaustive list of examples of cell types that have been successfully grown in Alvetex 3D cell culture. Many of these have been grown by REPROCELL scientists (see Alvetex protocols), while others have been reported in scientific journals (see Alvetex publications).

Alvetex compatibility with biology techniques 

Alvetex is compatible with a wide range of cell biology techniques. Following are some examples.

Imaging

Imaging reveals the integrity of in vivo-like structure and organization.

Changes in cellular morphology and function limit the value of cells grown in 2D cell culture. 3D culture systems enable cells to form more complex structures. Various models have been developed to create 3D skin constructs in vitro, including raft cultures. These methods are often technically challenging, involve multiple steps, show poor reproducibility and are difficult to practice routinely.

Alvetex provides an alternative method for 3D growth of keratinocytes, enabling reproduction of natural in vivo structures including the development of the stratum corneum, an essential component of the epidermal barrier. Skin constructs generated on Alvetex can then be used for drug and allergen penetration studies as well as assessment of barrier function.

Above: Histology images showing a full thickness skin construct grown by co-culturing human dermal fibroblasts inside Alvetex Scaffold and human keratinocytes on top, thus replicating both the dermal and epidermal compartments. Note that the presence of a stratum corneum is also evident. Validation of dermal and epidermal structure in full-thickness human skin equivalents. A: Representative photomicrographs of haematoxylin and eosin (H&E) stained Alvetex Scaffold seeded with human dermal fibroblasts after culture in Media A for 18 days. B & C: Representative photomicrographs showing H&E stained 35 day full-thickness human skin equivalents at 20× and 10× magnification respectively.*

* Data generated during a collaborative project between Reinnervate Ltd and Newcastle University – data now published in the following paper: A Novel Fully Humanized 3D Skin Equivalent to Model Early Melanoma Invasion. Authors: D.S. Hill, N.D. Robinson, M. P. Caley, M. Chen, E.A. OʼToole, J.L. Armstrong, S. Przyborsky and P.E. Lovat. Mol Cancer Ther. 2015 November ; 14(11): 2665–2673. doi:10.1158/1535-7163.MCT-15-0394.

Coating

Alvetex can be coated with extracellular matrix (ECM) proteins and other reagents commonly used to treat cell culture substrates, notably: Collagen I; Collagen IV; Fibronectin; Laminin; Poly-D-lysine; Poly-L-lysine; Poly-D-lysine and Laminin; Poly-L-orthinine and Laminin; Matrigel™; PuraMatrix™.

Explanting

Directly explanted cells can move into Alvetex directly from pieces of primary tissue or cell aggregates, migrating freely into the scaffold and spreading throughout its structure. Depending on cell type and characteristics, cells may proliferate as well as migrate. By enabling cells to be explanted in this way, Alvetex creates the opportunity for many different applications including tumor cell biology, separation of alternative cell types and establishing and maintaining 3D cultures de novo directly from primary sources, etc.

Above: Examples of cells from tissue pieces placed on top of Alvetex Scaffold migrating into the structure of the scaffold. A: A neural aggregate generated on a low-adherence plate before transfer to Alvetex Scaffold shows extensive neurite elongation within the thickness of the scaffold. B: Cells from an embryonal carcinoma aggregate readily invade Alvetex Scaffold.

Freshly-obtained intact tissues can also be maintained directly on Alvetex Strata for improved adherence and stability during imaging.

Above: Time lapse imaging of spinal cord tissue slice maintained on Alvetex Strata demonstrates minimal tissue drift over a period of 24 hours.

(Images courtesy of Kieran McDermott, University of Cork.)

Transfection

Various types of cells can be transfected using Alvetex 3D cell culture.

In collaboration with Mirus Bio, methods have been developed that enable the transfection of cells grown in Alvetex 3D culture. Common cell types (CHO-K1, HeLa, HepG2, MCF-7 and NIH-3T3) were seeded at optimized cell densities in 12 well Alvetex Scaffold 3D plates and adapted to 3D culture conditions for 48 hours. After adaptation, cells were transfected with a novel Mirus Bio formulation combined with a plasmid encoding firefly luciferase at the reagent-to-DNA ratios indicated beneath the bars. Luciferase activity was measured 24 hours post-transfection using a conventional assay. High expression was detected in all cell types demonstrating the efficiency of the Mirus Bio TransIT® 3D Transfection Reagent (MIR 5804) when used with Alvetex Scaffold 3D culture plates.

3D transfection of multiple cell types.

Above: Fibroblasts grown in 3D using Alvetex Scaffold were successfully transfected with a GFP construct and imaged using confocal microscopy. In brief, cells were transfected with the new Mirus Bio Transfection Reagent for 3D transfection at a reagent-to-DNA ratio of 3:1 using a GFP-expressing plasmid. Cells were seeded at 48 hours prior to transfection, and the cultures were fixed 24 hours post-transfection. Cells were imaged using a confocal microscope (Zeiss LSM510). The data shows a 40 μm integrated stack of multiple images as viewed from above the intact 3D culture. The position of all the cell nuclei are visualized with Hoechst 33342 (blue) and the positively transfected cells express GFP (green).

Co-culture

Advancing from single cell mono-cultures and co-cultures in conventional 2D models, Alvetex Scaffold provides the next step towards replicating the in vivo environment by providing the architecture necessary for 3D cell culture in vitro. Alvetex’s extremely high porosity allows cells to penetrate, grow and proliferate throughout the material for highly effective and reproducible 3D cell culture. Cells are freely able to form complex interactions with adjacent cells and receive and transmit signals, enabling a more natural environment to foster the native architecture found in tissues.

As well as being able to study single cultures in 3D, Alvetex Scaffold provides a support that enables the co-culture of more than one cell type. The structure of tissues is often comprised of discrete layers of distinct cell types. Growing different cell types in 3D, inside and on the surface of Alvetex Scaffold, enables users to re-create such tissue structures in vitro. A variety of cell co-culture scenarios can be set up to study different cell-cell interactions, according to the requirements of the cells and the dynamics under investigation.

Key benefits and applications:

• Study interactions between distinct cell types in 3D culture
• Recreate in vivo tissue morphology
• Recreate specific niche environments for disease modelling or drug testing
• Customize co-culture setup to suit the cell types involved

Several alternative approaches for co-culture design can be utilised. Alvetex Scaffold is a versatile technology that enables users to create co-culture models in many different ways.

Assembly of Co-culturing Experiments

Key to image parts:

Assembly option 1 – 3D co-culture in multiwell plate or well insert:

Description: Different cell types cultured together within the same scaffold.

Application: Emulate the structure of a tissue comprised of more than one cell type.

Assembly option 2 ‐ 3D / 2D co-culture in multiwell plate and well insert combined:

Description: Different cell types cultured together within the same scaffold within a well insert.

Application: Approach used to study the secretion of factors and signaling molecules.

Assembly option 3 ‐ 3D co-culture in multiwell plate and well insert combined:

Description: Two independent 3D cultures. Contact is via medium – communication via paracrine factors.

Application: Approach used to study the secretion of factors and signaling molecules.

Assembly option 5 ‐ 2D / 3D co-culture in multiwell plate:

Description: One 2D (monolayer) and one 3D culture layered in direct contact with one another

Application:

• Study the direct interaction of cells in contact with one another
• To establish layers of alternate cell types in 3D to mimic tissue structures
• To investigate invasion and migration of different cell types amongst each other

Exmples of Co-culturing Experiments

Above: Co-culture of glial and neural cells to model brain tissues. Brightfield micrograph showing the structure of a human stem cell-derived neurosphere co-cultured for 7 days with U118-MG glial cells on Alvetex Scaffold presented in the 12-well insert in 12-well plate format.

Above: Cell invasion into Alvetex Scaffold. Brightfield micrograph showing the structure of SW480 colon adenocarcinoma cells co-cultured for 7 days with established 3D cultures of 3T3 fibroblasts on Alvetex Scaffold.

Above: Enhanced cell function with hepatocyte and endothelial cell co-culture. The activity of CYP3A4 in upcyte® hepatocytes cultured on a 2D plate and on Alvetex Scaffold presented in both a 12-well plate and 6-well insert formats. Hepatocytes were grown as mono-cultures and also co-cultured with upcyte® micro-vascular endothelial cells for 10 days. For further details please visit www.medicyte.com.

Above: Co-culture of adipose tissue-derived stem cells with endothelial cells influences their differentiation. Adipose tissue-derived stem cells (ASCs) and endothelial cells (b.END-3) were cultured independently and as co-cultures in Alvetex Scaffold 12 well plate format for 3 days. Data from 3 sample replicates. VEGF levels normalized to the number of cells at the time of seeding, expressed as pg/mL per 10 cells. For further details please refer to Neofytou, E.A., et al. Adipose tissue-derived stem cells display a proangiogenic phenotype on 3D scaffolds. J Biomed Mater Res A. 2011; 98(3): 383-93.

Alvetex Compatibility with Downstream Applications

Alvetex is compatible with a wide range of downstream applications, such as:

Sectioning and Counterstaining

Unlike other 3D cell culture supports, Alvetex can easily be processed like a standard tissue sample. Frozen and paraffin embedded samples can be sectioned and stained to reveal the native cellular structures inside Alvetex. In this example, cells have been fixed in 4 % paraformaldehyde, embedded in paraffin wax, sectioned (7 μm) before staining with H&E and cover-slipped.

Immunocytochemistry

Ki67 DAPI Phase Immunocytochemistry used to visualize the expression of specific protein markers. In this example, cell cultures have been fixed in 4 % paraformaldehyde, embedded in paraffin wax and sectioned (10 μm). Antigen retrieval followed by immunocytochemical analysis with the proliferation marker Ki67 (green) and the nuclear stain DAPI (blue) was performed following standard immunocytochemical methods.

Scanning Electron Microscopy

Visualization of the structure of 3D cultures in Alvetex Scaffold is made possible using scanning electron microscopy (SEM). Samples are prepared in the same manner as would normally be used for tissues. In this example, skin, cells have penetrated throughout the scaffold and some have stratified on the surface (lower right corner).

Transmission Electron Microscopy

The ultrastructure of cells grown in Alvetex Strata can be analyzed by standard transmission electron microscopy (TEM). At high magnification, cellular structures such as these specialized bile canalicular cell protrusions are readily visualized.

Gene Expression Analysis

Isolation of nucleic acid from rat primary hepatocytes grown in Alvetex and conventional 2D cultures. RNA quality was determined by the RIN (RNA Integrity Number) and showed that the quantity and quality of RNA isolated from cells grown on Alvetex Scaffold was the same if not better than that isolated from standard 2D cultures. Data generated in collaboration with LGC (unpublished).

Protein Expression Analysis

With standard lysis protocols, total protein can be efficiently isolated from cells growing inside Alvetex. This allows for more biologically relevant protein expression analysis experiments to be carried out. Here we showed the increase over time of involucrin (Inv) expression in maturing keratinocytes by western blot analysis.

Isolation of Cells from Alvetex

With standard lysis protocols, total protein can be efficiently isolated from cells growing inside Alvetex. This allows for more biologically relevant protein expression analysis experiments to be carried out. Here we showed the increase over time of involucrin (Inv) expression in maturing keratinocytes by western blot analysis.

Biochemical Assays

Cells growing in 3D inside Alvetex can be studied using typical biochemical assays such as cell viability assays, apoptosis assays, cell proliferation assays etc. Here we show the measurement of cell viability using a standard MTT assay.

Perfused 3D Cell Culture

Alvetex perfusion plates enable dynamic circulation of culture media through wells into which Alvetex well inserts can be suspended. These systems can be used to create complex co-cultures, multi organ systems and to study paracrine effects.

Above. A: Alvetex Scaffold (scanning electron micrograph image) B: Alvetex Scaffold 6 well inserts. C: Cells grown in Alvetex maintain their 3D structure and form interactions with adjacent cells, resulting in a tissue-like structure. D: Alvetex well inserts fit inside the perfusion plate wells, enabling the flow of culture medium above and below the 3D culture.

Advanced 3D Tissue Bioengineering

The Alvetex Advanced 3D Tissue Bioengineering System is designed for scientists who need biologically relevant, reproducible models such as skin for testing pharmaceuticals , cosmetics , chemicals , and devices .

Above: Bioengineered full thickness human skin model (sample prepared for histology and stained with H&E). The white structures within the dermal compartment are Alvetex Scaffold.

See also REPROCELL's Alvetex 3D Bioengineered Tissue Model Assay Services.

Alvetex Plasticware Ranges

There are currently two ranges of Alvetex plasticware (bespoke plasticware containing Alvetex membrane).

Alvetex 3D Cell Culture Systems:

• A platform for routine 3D cell culture, designed for cell biologists who want to move beyond standard 2D monolayer cell cultures to 3D.
• Designed for routine mammalian and human 3D cell culture and basic co-culture models.
• Also suited for labs transitioning to 3D without full tissue engineering complexity.

Alvetex Advanced 3D Tissue Bioengineering System:

• Advanced platform for 3D tissue bioengineering with multiple cell types.
• Designed for building organotypic tissue models (e.g., skin, mucosa, barrier tissue).
• Designed for Pharmaceutical / cosmetic R&D teams that need reproducible models for barrier‐function measurements (TEER, water loss, permeability), topical application, and device/tissue interface studies.

Note: all products are supplied in sterile gamma irradiated packs.

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