The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.
Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.
The extracellular matrix (ECM) is comprised of a complex 3D network of proteins, glycoproteins, and polysaccharides1. Once regarded as a predominantly structural framework, it is now well recognized that the ECM incorporates a diverse array of bioactive molecules with important functional roles2. Cell-ECM interactions can direct cell survival, adhesion, migration, proliferation, and differentiation3. While the major classes of ECM macromolecules are generally well conserved across tissues and species, each tissue has a unique matrix composition and architecture4. Overall, the tissue-specific ECM provides an instructive microenvironment that mediates function from the subcellular to the tissue/organ scale5.
Due to the critical role of the ECM in mediating cellular function, there has been increasing interest in the development of ECM-derived bioscaffolds for applications in tissue engineering and regenerative medicine. In particular, the method of decellularization has been extensively explored as a means of obtaining ECM from a wide range of tissues for use as a scaffolding material for tissue regeneration and cell delivery5,6,7. Decellularization typically involves a series of mechanical, chemical, and/or biological treatment stages targeted at removing cells and cellular components, while ideally causing minimal alterations to the 3D structure and composition of the ECM8. Through surveying the literature, various decellularization protocols can be identified for virtually every tissue in the body7.
While decellularized tissues can be used directly as implantable scaffolds or 3D cell culture substrates, cellular infiltration may be limited in tissues with a dense ECM structure9. Further, the natural heterogeneity in the ECM may cause variability in cell attachment and distribution within the decellularized matrices, which could potentially impact the cellular response10. Overall, while promising for some applications, applying decellularized tissues in their intact form offers limited versatility in terms of tuning scaffold properties including shape, porosity, and stiffness, as well as the mode of delivery for in vivo applications.
To circumvent these limitations, numerous research groups are applying further processing methods to generate customized scaffold formats using decellularized tissues as a base material. In the simplest form, this may involve cryomilling the decellularized tissues to generate injectable tissue-specific ECM particles11. These ECM particles may be incorporated as a cell-instructive component in composite scaffolds with other biomaterials, such as in situ crosslinking hydrogels12,13,14. In addition to mechanical processing, decellularized tissues can also be subjected to enzymatic digestion with proteolytic and/or glycolytic enzymes to fabricate ECM-derived hydrogels, foams, microcarriers, and coatings15,16,17, as well as to synthesize bioinks for 3D printing18.
In addition to tissue-engineering applications, ECM-derived bioscaffolds hold great potential for the generation of higher fidelity in vitro models for biological research. There is a significant need to develop 3D cell culture systems that better recapitulate the native cellular microenvironment19. Most in vitro cell culture studies to date are conducted on tissue culture polystyrene (TCPS), which has little correlation with the biologically complex and dynamic cellular milieu found within living tissues20. While convenient for studying cellular interactions within a controlled environment, culturing cells on these simplified rigid 2D substrates alters cell attachment and morphology, as well as both cell-cell and cell-ECM interactions21,22. The cellular adaptations observed on 2D TCPS can impact intracellular signaling pathways that regulate diverse cell functions including survival, proliferation, migration, and differentiation, raising questions of the relevance of 2D studies in modelling in vivo systems23. There has been increasing recognition that cellular behavior can vary greatly in 2D versus 3D systems24, and that biochemical and biomechanical signaling with the ECM are key mediators of cell function25. Many groups have attempted to overcome the limitations of established 2D systems by coating TCPS with ECM components such as collagen, laminin, and fibronectin. While these strategies can improve cell attachment and may alter cellular responses, these models remain limited by their 2D configuration that does not mimic the complex spatial organization or biochemistry of the native ECM26,27.
Our bioengineering laboratory has been interested in the development of ECM-derived bioscaffolds as substrates for 3-D cell culture and tissue-engineering applications. In particular, we have pioneered the use of decellularized adipose tissue (DAT) as a scaffolding platform for adipose regeneration28. Moreover, we have established methods for synthesizing 3D microcarriers and porous foams using DAT digested with the proteolytic enzyme pepsin or glycolytic enzyme α-amylase29,30,31. Notably, we have demonstrated across all of these scaffold formats that the adipose-derived ECM provides an inductive microenvironment for the adipogenic differentiation of human adipose-derived stem/stromal cells (ASCs) in culture. More recently, we extended our fabrication methods to generate 3D porous foams from α-amylase-digested porcine decellularized left ventricle (DLV) (decellularization methods adapted from Wainwright et al.32), and showed that they provide a supportive platform for inducing early cardiomyogenic marker expression in human pericardial fat-derived ASCs31.
This article describes in detail the methods for synthesizing non-chemically crosslinked 3D porous foams and microcarriers derived purely from α-amylase-digested ECM for use as biologically complex 3D in vitro cell culture substrates and as biomaterials for tissue regeneration. In theory, any ECM source containing high molecular weight collagen may be used as the starting material for these techniques. To demonstrate the flexibility of this approach, the methods have been applied to generate tissue-specific bioscaffolds using human DAT, porcine decellularized dermal tissue (DDT)8, and porcine DLV as representative examples. Figure 1 provides a visual overview of the fabrication process for the ECM-derived foams and microcarriers.
Figure 1. Overview of the Method for the Production of the Tissue-specific ECM-derived Foams and Microcarriers. 1. Decellularized tissues, prepared following established decellularization protocols, can be used for tissue-specific ECM-derived bioscaffold fabrication. Macroscopic images are shown of hydrated human DAT (prepared as described in Flynn 201028), porcine DDT (prepared as described in Reing, J. E., et al. 20108), and porcine DLV (prepared as described in Wainwright et al. 201032), as representative examples of ECM sources that can be used as starting materials. Scale bars represent 3 cm. 2. The decellularized tissues are lyophilized, and then 3. mechanically minced. Scale bars represent 1 cm. 4. The minced ECM can then be cryomilled, which is optional for foam fabrication, but required for microcarrier synthesis. Scale bar represents 3 mm. 5. The minced or cryomilled ECM is then digested with α-amylase and homogenized to create a homogenous ECM suspension. Scale bar represents 1 cm. 6a) For foam fabrication, the ECM suspension is transferred into a user-defined mold, frozen, and lyophilized to generate a porous 3D scaffold with a well-defined geometry. Scale bar represents 1 cm. 6b) For microcarrier fabrication, the cryomilled ECM suspension is electrosprayed to generate discrete spherical microcarriers. Scale bar represents 2 mm. 7. The foams and microcarriers can then be gradually rehydrated and seeded with cells. Representative images are shown of human ASCs (viable cells=green) seeded on a DAT foam (left) and DAT microcarrier (right). Scale bars represent 100 µm. Please click here to view a larger version of this figure.
1. Decellularized Tissue Processing
2. ECM-derived Foam Fabrication
3. ECM-derived Microcarrier Fabrication via Electrospraying
NOTE: An overview of the electrospraying set up is shown in Figure 2.
Figure 2. Overview of the Electrospraying Apparatus used in Microcarrier Fabrication. A: Image showing the arrangement of the key electrospraying equipment including the syringe pump and high voltage power supply, as well as the positioning of the needle relative to the Dewar of liquid nitrogen. B: Electrospraying schematic, including the recommended ranges for the voltage, infusion rate, and distance. Please click here to view a larger version of this figure.
4. Preparing Foams and Microcarriers for Cell Culture
In the current study, we have fabricated ECM-derived foams and microcarriers using human DAT, porcine DDT, and porcine DLV as representative examples demonstrating that the techniques can be applied to generate tissue-specific bioscaffolds using a variety of decellularized tissues as ECM sources (Figure 1). For both foam and microcarrier fabrication, the Nishihara technique of collagen solubilization with the glycolytic enzyme α-amylase37 was adapted to generate a viscous ECM suspension from the decellularized tissue starting materials, which is used to synthesize the bioscaffolds through controlled freezing and lyophilization procedures.
To fabricate the foams, the decellularized tissues can be processed through either mechanical mincing or cryomilling to increase the surface area prior to enzymatic digestion. Following α-amylase treatment and homogenization, the resultant ECM suspension is dispensed into a user-defined mold, which is then frozen and lyophilized. The scaffolds can be stored stably in a dry state for an extended period of time. Prior to use in cell culture studies, the lyophilized scaffolds must be subjected to a controlled rehydration process that yields porous and highly-hydrated homogeneous foams derived from pure ECM (Figure 3A). If the foams are rehydrated too quickly, rapid swelling may damage delicate structural features, resulting in loss of integrity and structural collapse. In general, the foams will retain the shape defined by the original mold following rehydration and are stable without the need for chemical crosslinking. Figure 3B shows representative scanning electron microscope (SEM) images of the DAT, DDT, and DLV foams fabricated with cryomilled ECM at a concentration of 35 mg/mL and a freezing temperature of -80 °C.
Figure 3. Representative Images of the DAT, DDT, and DLV Foams Fabricated with Cryomilled ECM Suspensions at a Concentration of 35 mg/mL and Frozen at -80 °C. A: Macroscopic view of the DAT, DDT, and DLV milled foams synthesized in a 48-well tissue culture plate mold following rehydration. Scale bars represent 1 cm. B: SEM images of the DAT, DDT, and DLV foams showing a homogeneous porous ultrastructure. Scale bars represent 100 µm. Please click here to view a larger version of this figure.
The cryomilled ECM suspensions can also be used to generate pure ECM-derived microcarriers via electrospraying techniques (Figure 2). For each ECM source, the suspension concentration, needle gauge, infusion rate, and voltage can be tuned to generate discrete spherical microcarriers ranging from 350 – 500 μm in diameter following controlled rehydration (Figure 4A). While the microcarriers can be stored in a lyophilized state, it is recommended for ease of handling that they are dispensed or sieved to a desired size range following resuspension in ethanol. SEM imaging suggests that the microcarrier ultrastructure can vary depending on the decellularized tissue source (Figure 4B).
Figure 4. Representative Images of the DAT, DDT, and DLV Microcarriers Fabricated with Cryomilled ECM Suspensions at a Concentration of 35 mg/mL, an Infusion Rate of 0.5 mL/min, and an Applied Voltage of 20 kV. A: Macroscopic view of the hydrated DAT, DDT, and DLV microcarriers. Scale bars represent 4 mm. B: SEM images of the DAT, DDT, and DLV microcarriers showing that the ultrastructure and size can vary depending on the ECM source. Scale bars represent 100 µm. Please click here to view a larger version of this figure.
The mechanical properties of the scaffolds are dependent on the ECM source, the method of processing applied (i.e. mincing versus cryomilling), the ECM suspension concentration, and the freezing temperature. In general, the scaffolds are soft and compliant, with Young's moduli in the range of 1 – 5 kPa reported for the DAT (50 & 100 mg/mL)29 and DLV foams (20 – 50 mg/mL)31, and < 1 kPa for the microcarriers. The cryomilled foams are typically softer than the minced foams due to their more disrupted nature. Specialized mechanical testing systems equipped with highly-sensitive load cells are needed for accurate characterization of the compressive properties. Typically, the foams and microcarriers would have lower moduli than the native decellularized tissues due to the additional processing steps involved in fabrication including enzymatic digestion and homogenization, as well as the non-covalently crosslinked nature of the scaffolds. However, this can vary depending on the tissue of interest. For example, in previous work, we found that minced DAT foams fabricated at high ECM concentrations (100 mg/mL) had similar moduli to native adipose tissue29.
The rehydrated ECM-derived foams and microcarriers can be seeded with cells under static or dynamic culture conditions to provide a cell supportive platform for in vitro cell culture studies and/or in vivo cell delivery. While the scaffolds generally support cell attachment, the seeding efficiency will depend on the ECM source, the scaffold geometry and porosity, and the cell type of interest. As such, the seeding methods and densities will require optimization depending on the user-defined conditions. For the foams described above, we recommend a starting cell concentration in the range of 0.25 – 1 x 106 cells/scaffold, with dynamic seeding on an orbital shaker to enhance cell infiltration. For the microcarriers, we suggest an initial seeding density in the range of 25,000 – 50,000 cells/mg of microcarriers, with seeding performed under dynamic conditions in a spinner culture flask11. The images shown at the bottom of Figure 1 represent human ASCs seeded on a DAT foam (left) or DAT microcarrier (right, prelabeled with an amine-reactive dye and appearing blue13), as visualized by confocal microscopy using a fluorescent cell viability stain (live cells = green, dead cells = red; Scale bars represent 100 µm). Confocal microscopy can be used to visualize cells seeded on the surface of foams up to 2 mm in thickness, but visualization into the central regions of the scaffolds is limited by their opaque nature. However, the foams and microcarriers can be treated as tissues and paraffin-embedded or cryo-sectioned for histological and immunohistochemical analyses to visualize cell distribution and marker expression.
In general, bioscaffolds derived from decellularized tissues can more closely approximate the complex 3D composition and structure of the ECM in the native cellular microenvironment as compared to synthetic scaffolds or standard culture models based on 2D TCPS. As previously discussed, cell-ECM interactions are critically important in mediating cellular behavior both in culture and in the body1. Recognizing that the biochemical, biophysical, and biomechanical properties of the ECM are unique to each tissue, there is increasing evidence to support the rationale for applying tissue-specific approaches in the design of biomaterials for tissue engineering, as well as in the development of more physiologically relevant culture models for in vitro experiments20. Utilizing decellularized tissues as a starting material, our methods can incorporate the complex composition of the tissue-specific ECM within more customizable scaffold formats. While the mechanical and enzymatic processing steps will result in a loss of the native ECM ultrastructure, previous studies with DAT have demonstrated that the instructive effects of the adipose-derived ECM are conserved in these scaffold formats, suggesting that the bioscaffold composition is a key mediator of cell function11,29. A significant advantage to using the ECM-derived foams and microcarriers as cell culture substrates as compared to the intact decellularized tissues is that they are more homogeneous, which can improve uniformity in cell distribution and cell-cell/cell-ECM interactions.
The methods described here can be utilized to generate a broad array of tissue-specific bioscaffolds for use in cell culture and tissue-engineering applications. For example, in addition to the DAT, DDT, and DLV, our lab has successfully applied these techniques to generate 3D porous foams using decellularized bone, cartilage, nucleus pulposus, and annulus fibrosis, as well as commercially-available, insoluble collagen derived from bovine tendon. From an in vitro perspective, these bioscaffolds could be used as a basis for higher-fidelity 3D culture models for investigating cellular biology, physiology or disease pathology38, as bioactive substrates in high-throughput drug screening platforms39, or as instructive matrices for stem cell differentiation40,41. DAT, DDT and DLV foams fabricated at concentrations of 25 – 50 mg/mL are stable in long-term in vitro culture (tested up to 28 days). Further, all three types of microcarriers can support cell attachment and proliferation under dynamic conditions in a low-shear spinner culture system (10 – 15 rpm) for at least 2 weeks. For in vivo applications, the biocompatible and biodegradable ECM-derived foams and microcarriers hold promise as off-the-shelf products to stimulate constructive tissue remodeling and regeneration11,29. Further, the cell-adhesive scaffolds could be used as cell therapy delivery systems42,43. As an example, DAT foams were shown to promote angiogenesis and adipogenesis when seeded with allogeneic ASCs and implanted subcutaneously in an immunocompetent rat model29. Relative to the intact DAT, the more highly processed DAT foams degraded much more rapidly, with a 50% reduction in volume noted at 3 weeks as they became integrated with the host tissues, and almost complete resorption by 12 weeks. However, the foams also induced a more potent angiogenic response, suggesting that the enzyme-digested ECM had unique pro-regenerative effects. Similarly, the ECM-derived microcarriers could be used as in vitro cell culture substrates within dynamic culture systems and as injectable cell delivery vehicles11,30,44. More specifically, the small diameter and large surface area of the microcarriers could enable the delivery of a large quantity of cells in a small volume, while providing a matrix that may help to support cell viability and increase cell retention at the site of injection30. Prior to use in any living system, it is critical to ensure that the source ECM is substantially devoid of antigenic cellular components and/or potentially cytotoxic decellularization reagents that could trigger a negative host response7.
The proteolytic enzyme pepsin is commonly used in the preparation of ECM-derived hydrogels15. Pepsin is a non-specific protease that will digest collagen and other ECM proteins into small fragments45. While hydrogels fabricated from pepsin-digested ECM have been reported to have cell-instructive effects, a limitation is that these materials tend to be extremely mechanically weak46. In our initial development of the DAT microcarriers, we utilized a composite approach in which pepsin-digested DAT was combined with alginate and added dropwise into CaCl2 to form spherical beads30. The beads were subsequently photo-crosslinked and the alginate was extracted using sodium citrate. In addition to the requirement for chemical crosslinking, a key limitation was that the microcarriers fabricated with this approach had poor stability below a size range of 900 – 950 µm30. In place of pepsin, the methods presented here utilize a more mild digestion of the ECM with the glycolytic enzyme α-amylase, which is postulated to cleave carbohydrate groups from the telopeptide regions of collagen, thereby increasing solubility in acetic acid37. This approach enables the isolation of highly polymerized collagen that can be used to generate pure ECM-derived foams and microcarriers without the need for chemical crosslinking or other additives. These bioscaffolds are stabilized through physical interactions and hydrogen bonding between well-preserved collagen fibrils, similar to the collagen in the native ECM microenvironment.
The foams are a highly flexible platform that can be fabricated in a wide range of geometries depending on the specific mold selected. For cell culture studies, the foams may be cast directly in TCPS well plates, to form coatings or 3D scaffolds of varying thickness. To fabricate 3D foams with very uniform surfaces, it is recommended that a custom mold is designed that can be sealed on both sides with plastic or glass slides. Either minced or cryomilled ECM can be used to synthesize the foams. In general, we have found that the cryomilled foams tend to be macroscopically softer and have a more disrupted ultrastructure at lower concentrations31,36. Depending on the tissue source, the additional mechanical processing steps may cause alterations in the ECM composition that could impact cell function. For example, in our previous work, laminin was detected in minced DLV foams, but not cryomilled DLV foams31. In contrast, collagen I, collagen IV, laminin, and fibronectin were detected in both minced and cryomilled DAT foams36. In addition to the mechanical processing steps, the porosity and pore size of the foams can be tuned to some extent by varying the ECM suspension concentration and the freezing temperature47. In general, lower concentration foams (~ 10 – 15 mg/mL) are qualitatively more porous, but may contract rapidly and have poor stability in long-term culture31,36. Similarly, a slower freezing rate, typically achieved by a higher freezing temperature, can result in larger pores in the foams due to the size of the ice crystals formed during fabrication29. All of these parameters may influence cell interactions with the materials, including attachment, infiltration, and remodeling. For example, cell growth on foams that are fabricated with higher ECM concentrations may be limited to the surface regions, particularly with minced ECM sources and under static culture conditions36.
For the microcarriers, the key parameters that can be tuned are the ECM suspension concentration, needle gauge, and applied voltage, with higher concentrations typically yielding microcarriers that are more stable under long-term dynamic culture. Following the initiation of electrospraying, the ECM suspension droplets should quickly fall into the center of the flask, towards the direction of the aluminum foil collector. To prevent aggregation, it is important that the beads contact the liquid nitrogen prior to the foil. The distance between the needle and the surface of the liquid nitrogen can be adjusted to meet these requirements. It is important to note that optimization may be required depending on the properties of each specific ECM source, in particular in selecting the concentration range that will generate stable bioscaffolds. Another key factor is the decellularization protocol that is used to generate the starting materials, as decellularization methods that degrade the ECM or the presence of residual reagents (e.g., surfactants) may negatively impact the stability of the resultant foams and microcarriers. If challenges are encountered with bioscaffold stability, options that can be investigated include using a more gradual rehydration process, increasing the ECM suspension concentration, and exploring minced versus cryomilled ECM. Should all of these options fail to resolve the issue, it may be necessary to explore alternative decellularization protocols or ECM sources.
To ensure reproducibility during scaffold production, special care must be taken at certain steps in the protocol. When cryomilling the decellularized tissues, it is recommended that milling be conducted immediately after lyophilization in a dry environment to reduce the likelihood of particle aggregation due to the absorption of moisture from the environment. During microcarrier fabrication, it is suggested that the suspension is electrosprayed in small batches, with a maximum volume of 3 mL, to avoid issues with sample cooling that can result in clogging of the needle. Further, it is essential that the microcarriers are not permitted to thaw after the electrospraying process. To maintain their spherical geometry and mechanical stability, the microcarriers should be collected from the liquid nitrogen, transported in a liquid nitrogen-filled container, and immediately lyophilized. Finally, for both the foams and microcarriers, it is critical that the rehydration steps are performed slowly over a period of multiple days. Rapid rehydration can result in structural collapse on the macro- and/or micro-scale. Further, rehydration must occur slowly to prevent the formation of small air bubbles within the scaffold, which can require a significant amount of time to degas under light vacuum.
In conclusion, the methods presented in this paper can be used to fabricate a diverse array of tissue-specific foams and microcarriers comprised of pure, non-chemically crosslinked ECM. An advantage for biological researchers is that the bioscaffolds are easy to handle and can be processed similarly to tissues when performing analyses with techniques such as histology, immunohistochemistry, or gene and protein expression assays. In addition, the ECM-derived scaffolds can be enzymatically degraded to extract seeded cell populations or can be used directly as biodegradable and biocompatible cell delivery vehicles. Overall, this flexible platform technology holds great utility for numerous applications including for 3D cell culture studies investigating cell function, as cell expansion substrates, and as pro-regenerative bioscaffolds.
The authors have nothing to disclose.
The Natural Sciences and Engineering Research Council (NSERC) of Canada and the Canadian Institutes of Health Research (CIHR) have provided funding for this work. The authors would like to acknowledge Dr. Amin Rizkalla for the use of his electrospraying system, the Nanofabrication Facility at Western University for use of SEM imaging equipment, the Mount Brydges Abattoir for the provision of porcine tissue samples, and Drs. Aaron Grant, Brian Evans, and Robert Richards for their clinical collaborations in support of this research.
Acetic acid, glacial | BioShop | ACE222.500 | |
Alligator clip leads | VWR | 470149-728 | |
Aluminium foil | Fisher Scientific | 01-213-101 | |
a-amylase | Sigma | 101074694 | from Aspergillus aryzae |
Analytical balance | Sartorius | CPA225D | |
Centrifuge | Thermo Scientific | 75004251 | With swinging bucket rotor for 15 and 50 mL centrifuge tubes |
Centrifuge tubes (15 mL) | Sarstedt | 62.554.205 | |
Centrifuge tubes (50 mL) | Sarstedt | 62.547.205 | |
Collagen from bovine achilles tendon (insoluble) | Sigma | C9879 | Or similar insoluble collagen source; Can be used as an alternative to decellularized tissues to fabricate the foams and microcarriers |
Cryomilling system | Retsch | 20.745.0001 | MM 400 |
Dessicator | Fisher Scientific | 8624426 | For lyophilized ECM and bioscaffold storage |
DMEM: F12 Hams | Sigma | D6421 | Used for proliferation media |
Dewar flask | Fisher Scientific | 10-196-6 | Low form; volume range of 250 – 500 mL |
Double distilled water | From Barnstead GenPure xCAD Water Purification System | ||
D-PBS | Wisent | 311425125 | |
ECM | Isolated from human adipose tissue, porcine dermis or porcine myocardium, as described in Flynn et al. 2010, Reing et al. 2010, and Wainwright et al. 2010 (ref # 28, 8, 32) | ||
Ethanol | Greenfield Specialty Alcohols Inc. | P016EAAN | Absolute |
Fetal bovine serum | Wisent | 80150 | Used for proliferation media |
Forceps | VWR | 37-501-32 | For transferring the foams |
Freezer (-20 °C) | VWR | 97043-346 | |
Freezer ( -80 °C) | Thermo Scientific | EXF40086A | |
Glass vials | Fisher Scientific | 03-339-26D | To store lyophilized cryomilled ECM |
Hand held homogenizer | Fisher Scientific | 14-359-251 | Speed: 8000 – 30,000 RPM |
Homogenizer accessories: saw tooth bottom generator probes | Fisher Scientific | 14-261-17 | 10 X 95 mm |
Liquid nitrogen | For electrospraying | ||
Lyophilizer | Labconco | 7750021 | FreeZone4.5 |
Milling chamber | Retsch | 02.462.0213 | 25 mL volume recommended |
Milling balls | VWR | 16003-606 | 10 mm diameter, stainless steel recommended |
18G needle | VWR | C ABD305185 | For dispensing ECM suspension into moulds |
Orbital incubator shaker | SciLogex | 832010089999 | Temperature controlled (37 °C) |
Penicillin-streptomycin | Life Technologies | 15140-122 | Used for proliferation media |
Pipet-Aid XP | Mandel Scientific | DRU-4-000-101 | |
Retort stand | VWR | 470019-526 | |
Retort stand clamp | VWR | 21573-606 | |
Safety-Lok Syringe | BD | 309606 | 3 mL luer lock syringe for microcarrier fabrication and dispensing ECM suspension |
Serological pipettes (10 mL ) | Sarstedt | 86.1254.001 | |
Serological pipettes (25 mL) | Sarstedt | 86.1685.001 | |
Sodium chloride | BioShop | 7647-14-5 | |
Sodium phosphate monobasic | BioShop | 10049-21-5 | |
Scoopula | VWR | 89259-968 | For collecting microcarriers |
Surgical scissors | VWR | 82027-590 | |
Syringe pump | VWR | 10117-490 | Microprocessor controlled |
High voltage power supply | Gamma High Voltage Research | ES30P-5W/DDPM | Capable of recommended 15 – 25 kV voltage range |
12-well plates | Fisher Scientific | 12565321 | For use as molds during foam fabrication; Other sizes or user-defined molds can also be selected |
Winged infusion set | Terumo | 22258092 | 30 cm tubing length, 25 G 3/4 " recommended; Other needle gauges can be used and may influence microcarrier diameter |