In vitropre-vascularisation of tissue-engineered constructs A co-culture perspective
Vascular Cell. 2014;
Received: 16 February 2014 | Accepted: 12 June 2014 | Published: 21 June 2014
Vascular Cell ISSN: 2045-824X
KeywordsCo-culture Vascularisation Tissue engineering Matrices
Researchers have two main options when vascularizing tissue-engineered constructs: either implant the construct
A key first decision in designing an
Endothelial and precursor cells
Endothelial cells are present in most tissues within the human body; however, their relative abundance and composition varies from tissue to tissue . A microarray study on the expression profiles of 53 endothelial cells showed distinct tissue-specific expression patterns in cells isolated from different blood vessels and microvasculature in the body . There are a wide variety of different types of endothelial cells used in the literature. Researchers seeking to model a particular biological system or disease state may choose to isolate them directly from the tissue of interest. The logic behind isolating cells from the tissue of interest is that the researchers will be able to isolate endothelial subpopulations specific to the microenvironment that they wish to recapitulate. However, from a tissue engineering perspective, isolating tissue-specific endothelial cells may not be a feasible strategy as retrieving these cells may require an invasive procedure, and in the case of major organs or tissues may not be a viable option. In order for a specific cell-based tissue engineering approach to be practical in a clinical setting, the source of cells needs to be (i) relatively abundant, (ii) readily available and (iii) pose a minimal to low risk to patient/donors. Examples of non-invasive cell sources include placental or umbilical cords which are commonly discarded as medical waste, and examples of minimally invasive procedures for isolation of endothelial cells include peripheral blood and skin biopsy [14–16].
It is important to remember that isolated primary cells are heterogeneous and contain a mix of different endothelial cell subpopulations. In 2004 Ingram et al. identified a novel cell hierarchy among endothelial cells found in human peripheral and umbilical cord blood based on clonogenic and proliferative potential . The endothelial lineage is believed to follow a similar hierarchical as myeloid and lymphoid lineages in which a primitive stem cell gives rise to proliferating progenitor cells, followed by the progression to terminally differentiated cells . Figure 1 shows the model of endothelial cell hierarchy based on proliferative and clonogenic potential, thus defining endothelial progenitors (EPC) as cells giving rise to high proliferative colonies with the capacity to form blood vessels upon transplantation. A further study identified a subpopulation of endothelial progenitor cells (EPC) within human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells (HAEC) . Both HUVEC and HAEC can be isolated from vessel walls and were previously thought to consist of only mature differentiated endothelial cells . The heterogeneous composition of isolated endothelial cells may affect the reproducibility of cell-based treatments and isolated cells may need to be sorted into individual cell populations.
The therapeutic potential of EPC subpopulations of endothelial cells has garnered a significant amount of interest within the research community in recent years. EPCs have been shown to have enhanced proliferative potential, are able to differentiate and give rise to all subsets of the endothelial cell lineage and have been shown to improve vasculogenic activity . Other studies have also shown that EPC have a higher survival rate
CFU-Hill are non-adherent PB-MNC that give rise to colonies after 5 days following depletion of adherent cells on fibronectin and CAC cells are adherent PB-MNC that attach to fibronectin or gelatin surface after 4–6 days of culture [24, 25]. Both CFU-Hill and CAC cells co-express CD31, VEGFR-2 and CD133 . CD133 is a hematopoietic stem cell (HSC) marker that is lost as cell differentiate . Peichev et al. hypothesized that CD133 may also be a marker for immature EPC populations . A later study by Case et al. in 2007 however showed that 99% of CD34+ VEGFR-2+ CD133+ cells also co-expressed CD45+, a common leukocyte antigen, not expressed by endothelial cells . In addition CD34+ VEGFR-2+ CD133+ cells also readily ingest bacteria and lacked the ability to form human vessels de novo
ECFC on the other hand are late outgrowth cells that form colonies on type 1 rat tail collagen following 14–21 days of culture . According to Ingram et al. ECFC more closely match the criteria of being a true EPC . Unlike CFU-Hill and CAC cells, ECFCs do not express haemopoetic markers such as CD133 and CD45 . ECFC are highly proliferative and have the potential to form both secondary and tertiary colonies on replating . ECFC are also capable of forming human vessels de novo
The findings from these studies highlight the striking differences between the different classes of putative EPCs and have important implication for their therapeutic application. Although CD34+ VEGFR-2+ CD133+ cells cannot initiate vessel formation de novo
Another major limitation of ECFCs is their rarity within the human body. EPC only make up 0.01% of circulating mononuclear cells in peripheral blood . An alternative source of ECFC is umbilical cord blood or placental tissue. The number of endothelial cell colonies derived from umbilical cord blood was 15 times higher than from equivalent volumes (20 mL) of peripheral blood . The colonies formed by umbilical cord blood were also consistently larger than those isolated from peripheral blood . More recently, it has now become possible to also isolate ECFCs from the placenta tissue. From a single placenta (500-600 g) it is possible to isolated the same amount of ECFCs as found in 27 whole cord blood (60 mL) samples . Gene expression and functional studies have demonstrated that these cells are equivalent to umbilical cord blood derived ECFCs . The high number of ECFCs that can be derived from these tissues, that are commonly discarded, is opening new possibilities in tissue engineering, in particular in large defect applications that require high cell numbers.
An alternative strategy may be to expand isolated ECFC
Mature endothelial cells and endothelial cell clusters lack the ability to be expanded out into high numbers like their ECFC counterparts, however these endothelial cells are still capable of forming capillary-like structures. ECFC cells are responsive to angiogenic factors, have a high survival rate and are believed to play a key role in maintaining vessel wall integrity [18, 19]. Therefore the use of mature endothelial cells/endothelial cluster subpopulations to form an
Multipotent adult stem cells
Adult stem cells are multipotent cells that are capable of differentiating into a narrow range of different cell types . Recent advances in our understanding of stem cell biology and regulation have provided researchers with a range of novel tools and research strategies to guide cell fate both within and outside their traditional cell lineages. Bone marrow mesenchymal stem cells (BM-MSC) have been shown to readily differentiate into endothelial cells under angiogenic conditions. Differentiated BM-MSC express several endothelial markers
Pluripotent stem cells
In the past ten years there has been significant advancement in the fields of stem cell biology and iPS technology . These developments have had a tremendous impact on regenerative medicine and tissue engineering concepts. Unlike endothelial progenitor cells, pluripotent stem cells have the potential of differentiating into all three germ layers . The most recognizable and well characterized pluripotent cells are ESCs. A number of studies have been able to differentiate ESC into endothelial and associated mural cells [46, 47]. ESC-derived endothelial cells were shown to contribute to the construction of new blood vessels and improved blood flow in a hindlimb ischemia model . However ethical concerns surrounding the isolation of ESC limit the widespread application and adoption of this cell technology . Other technical limitations associated with the use of ESC include source availability, difficulty in separating out endothelial cells from undifferentiated ESC cell and the potential for ESC to form teratomas [49–51].
iPS cells are differentiated cells that have been genetically reprogrammed to return to a pluripotent stem cell state and therefore circumvent the need to source cells from embryos . A large body of research from the mid 1990s to early 2000s identified a number of key genes relating to maintenance and regulation of pluripotency in embryos and ESC [53–58]. In 2006 a seminal paper by Dr Shinya Yamanaka’s laboratory at Kyoto University screened 24 of these genes as candidates for inducing pluripotency in somatic cells and identified four factors, including Oct3/4, Sox2, c-Myc and Klf4, that were able to successfully produce iPS cells from mouse adult fibroblast cells . Since this study, a number of laboratories have been able to use Dr. Yamanaka’s technique to generate iPS cells from a variety of different cell types and species including humans . Once the cells have returned to a pluripotent state the cells can then be re-differentiated into endothelial and associated mural cells. Choi et al. were able to differentiate seven human iPS cell lines into endothelial (CD31+, CD34-) cells . The iPS-derived endothelial cells were shown to successfully form capillary-like structures on growth factor reduced matrigel in 2D. Another study by Samuel et al. was able to establish functional blood vessels long-term (280 days)
Pluripotent stem cells represent a potential universal cell source for endothelial cells, but the technology is still in the early stages of development and if researchers cannot rectify the issues associated with the cells, in particular the safety concerns, the technology will never be able to move beyond pre-clinical applications into clinical regenerative therapies.
Cells that are grown in conjunction with endothelial cells can have both a direct and indirect impact on the development of vascular networks in tissue-engineered constructs. Figure 2 highlights the role that these cells can have on capillary formation and maturation.
Assays for the functional assessment of vascularization
A number of
There are a number of parameters that require careful consideration when designing a co-culture system. Some conditions may be considered trivial, but can have important implications for the end tissue-engineered product.
In tissue engineering, scaffolds and matrices provide cells with support and structure to move from 2D tissue culture plate into a 3D microenvironment. The three main types of 3D scaffolds include; solid scaffolds, hydrogels (Figure 3A-B) or a combination of the two constructs (Figure 3C). Solid scaffolds are porous 3D structures, whilst gels are polymer networks that are expanded throughout their volume by fluid. Thus far, only hydrogels have been shown to form functional vascular networks with lumen
However, it is important to note the immersion of cells in a 3D environment by itself also will not lead to capillary formation
The mechanical properties of a construct can also play an important role in capillary formation. In literature the range of stiffness as measured by compression modulus for optimal tube formation in hydrogels varies between different gel types, however in all cases increased vasculature formation was associated with decreasing stiffness [92–95]. The phenotypic expression of other cell types on the other hand can increase with increasing stiffness. For example, in bone tissue engineering, stiffer gels can increase bone mineralization and expression of differentiation markers such as osteocalcin, osteopontin and alkaline phosphatase [96, 97]. The selection of optimal construct stiffness can be problematic if attributes (i.e. vascularisation and bone formation) are desired in the same construct, but inversely related. Researchers need to find a middle ground or alter another parameter in the system.
Fabrication of microchannels
A new wave of vascular constructs and designs are helping to speed up the tube formation process and assist with co-culture strategies. The process of capillary formation
Concepts from microfluidics can be utilized to engineer vasculature for tissue engineering. Microfabrication techniques, such as photolithography, can be used to etch micron sized open channels into a silicon master mould (Figure 3D) . A polymer of interest can then be poured into the mould in order to create a patterned surface. Once set the patterned layer can be removed and then bonded with a flat unpatterned layer of polymer to create a closed system. Different polymer layers can be fused using plasma treatment, temperature/pressure difference, or other polymer specific properties. These microfluidic constructs can then be further stacked and bonded to create larger 3D tissue engineered constructs. A major limitation of the layer bonding technique is the danger of leaks if the layers are not completely fused together.
Another microfluidic approach is to mould a biodegradable material into the shape of a vasculature network and then embed the construct within a biomaterial of interest (Figure 3E). The construct acts as a sacrificial material that degrades over time to leave hollow microchannels behind. This method can also be combined with additive manufacturing techniques to create complex 3D vasculature structures . Another benefit of biodegradable microfluidic channels is that unlike the layer bonding method the surrounding biomaterial formed is intact and there is a lower danger of leaks forming, however the drawback is that the technique doesn’t have the high resolution of other microfabrication techniques such as photolithography.
A final approach is to re-endothelialise decellularized whole tissue or organs using the existing vessel structures as templates (Figure 3F). Advances in decellularization processes in recent years have now made it possible to remove cells from tissue whilst retain the vital structure and bioactivity of the ECM . The organs and tissue can also be sourced from xenogenic tissues which are readily available. A limitation of this approach is that users are restricted to the layout of vascular structures in the tissue. There are also still unresolved concerns surrounding the antiginicity, immunogenicity and shelf life of decellularized organs .
Cell sheet technology
A final technique for developing tissue engineered constructs is cell sheet technology (Figure 3G). Cell sheet engineering is a non-scaffold based approach that uses temperature responsive cell culture surfaces to harvest intact cell sheets that can be stacked together to reconstructs 3D tissue . The temperature responsive culture surfaces are created by treating normal tissue culture plates with poly(N-isoproplyacrylamide)(PIPAAm) that can alternate between states of hydrophobicity and hydrophilicity . At temperatures higher than 32 degrees the substrate is hydrophobic and cells can attach to the surface and form a confluent layer. Lowering the temperature below 32 degrees causes the substrate to become hydrophilic and the cells sheet and ECM to detach from the surface. A gel coated plunger can then be used to manipulate and stack the cell sheets. The cell sheet technique has been used to effectively replicate tissue and organs, such as skin and cardiac tissue, both
The ratio between the different cell types in co-culture can influence cell characteristics, behavior and survival. In view of the available literature, no consensus exists on the optimal cell ratio of endothelial cells to tissue-specific cells for use in
A key characteristics to consider when selecting the ratio of cells to use in a co-culture system is the cells individual metabolic and proliferative potential. If the cells proliferative and metabolic activity differs significantly, depending on the duration of
The composition of culture media is one of the key points to consider when culturing cells
The use of serum is another important consideration in endothelial cell culture. Serum is a common media additive to support cell growth and viability
In a co-culture scenario each cell will have its own individual media requirements. In some cases the cells may utilise the same media, but often this will not be the case. If cells require different culture media, researchers need to optimize an appropriate media combination that offers acceptable viability, whilst promoting or maintaining desired cell phenotype. Most papers do not explain the decisions that led them to the selection of their chosen media or media combination, despite the critical role that this factor may play in the outcome of the study. The media mixture may depend on the ratio of each cell type used, the sensitivity of cells - one cell type being potentially more sensitive than another to alteration of media composition - and the purpose of the study.
The addition of supplements to media is another issue that has to be considered. Co-culturing cells together will change the expression profile of each cell type through paracrine signaling and cell-cell interactions. The endogenous factors secreted by the cells in the microenvironment may contribute to, or may inhibit, the usual effect of supplements in the medium. For example, Unger et al. showed that in monoculture of HDMEC, exogenous angiogenic factors, such as bFGF or VEGF, were required for microvascular formation . However, surprisingly when these components were added to co-cultures of HDMEC and osteoblasts, microcapillary formation did not occur. VEGF promotes endothelial cell motility, and in this case too much of this factor may be over stimulating the cells and destabilizing the network.
There may also be a potential significant difference between endothelial capillary formation that is driven by exogenous stimulus in the form of angiogenic supplements added to culture media versus endogenous angiogenic factors secreted by non-endothelial supporter cells in a co-culture system. The exogenous supplements are added as a single dose at the time of media change and their bioactivity will decrease with time depending on the initial concentration, stability of the supplement and relative uptake by the cells. Factors secreted by supporting cells in co-culture are released more steadily over time but as with the exogenous supplements the factors will be removed when the media is changed. Figure 4 shows a visual representation of this phenomenon. It is unclear how the difference between exogenous and endogenous stimulated capillary formation may influences the structural and functional aspects of the networks. In the end, the best way to determine the optimal medium composition may be experimentally by examining not only the proliferation or viability of each cell type but its impact on gene expression and cell phenotype.
There are two main types of seeding parameters researchers can modulate: temporal (seed simultaneously or sequentially) and spatial (seed on one construct or multiple constructs).
Seeding the cells in the matrix at the same time allows for a homogeneous mix of cells throughout the construct (Figure 5A). This is beneficial if cell-cell contact is important for cell function or if the cell types are naturally co-located with one another in the tissue of interest. Researchers can also use the same scaffold, but seed the cells at different times (Figure 5B). In addition to modifying the cell ratio, sequential seeding is beneficial if the cell proliferation rates differ significantly and there is the potential that the more proliferative cell type may take over the construct. Moreover, pre-seeding one cell type in the scaffold may help direct or bias the overall characteristics of the construct towards a particular phenotype or trait of interest. For example, Lyer et al. previously showed that following co-culture of EC, fibroblasts and cardiomyocytes in matrigel, the cells formed an organoid that mimicked cardiac structure and function, but the EC cells did not organize into capillary structures . However, a separate study from the same group seeded the EC cells first, followed by fibroblasts 24 hours later, and cardiomyocytes 48 hours later, which resulted in extensive cord formation in the end construct . Seeding the EC cells first may have provided the cells with time to form tubes unimpeded and the addition of fibroblasts may have provide the newly formed network with structural support before the addition of the cardiomyocytes. A difficulty associated with sequential seeding in the same construct is the requirement to incorporate cells in a solid scaffold or gel that has already been made. The construct would need to be either porous, include hollow microchannels, or require a chemoattractant to encourage cell ingrowth. In the case of Lyer et al., the organoids were thin microtissue .
Cells can also be spatially separated on different scaffolds (Figure 5C-D). As previously mentioned, the properties of scaffolds can influence the phenotype of the cells in the tissue-engineered constructs, which may be problematic if competing characteristics are required. A way around this problem is to place the cells in separate scaffolds with optimal properties for each desired cell trait. These scaffolds can be seeded either simultaneously or sequentially but again will depend on the proliferation rates of the cells and the desired cell traits in the construct. An added advantage of spatially separated constructs is that they can also be cultured in different medias before being combined (Figure 5Cii-Dii). This can overcome some of the possible problems associated with compromising on shared media conditions. The limitation of spatially separating the cells is that it allows only minimal cell-cell contact between the different cell types.
Bioreactor systems are commonly used within an
It has been long known that hypoxia can promote angiogenesis and vascularisation in tissue . An oxygen dependent homeostatic mechanism in the body ensures that all cells receive adequate blood supply . When tissues are exposed to a low oxygen environment they begin to express factors such as hypoxia-inducible factor 1(HIF-1) which promotes VEGF production . The VEGF then acts on the endothelial cells to promote cell migration and vascularisation. Researchers can mimic this cellular response by modulating oxygen tension in a controlled environment such as a incubator to control capillary formation in endothelial cells
Studies relating to the utilization of rotational bioreactors in co-culture systems have so far shown mixed results. Xing et al. immobilized scaffolds co-cultured with bone marrow stromal cells and endothelial cells on stationary needles in a spinner flask moving in a single direction on the x-axis . After a week in the bioreactor, extensive capillary networks formed within the scaffolds. However, in a study by Liu et al. that co-cultured EPC and MSC on immobilized scaffolds using a biaxial bioreactor which was rotating simultaneously on a perpendicular axes (X and Z), no vessel formation was observed in the dynamic system, but extensive vessel network formed under static conditions after a week of culture
Perfusion systems can be used to mimic the haemodynamic forces and pressures that occur naturally in the human body. Fluid flow in a bioreactor can be directed through the bulk of a construct, however in most cases it will be directed through hollow microchannels or pores within the construct, similar to those described in section 3.1, that have been pre-seeded with endothelial or perivascular cells. Several studies have shown that mechanical stimulation under peristaltic flow conditions can increase the production of ECM proteins, such as elastin and collagen, and improve the mechanical properties of the vessel or capillary as measured by burst pressure and resistance to shear stress . Mechanical stimulation by perfusion systems is critical in pre-conditioning vascular constructs prior to implantation.
There are a variety of cell sources for vascularized tissue constructs; however, endothelial cells are the one cell type that is ubiquitous in almost all systems. EC are heterogeneous in nature and contain a mix of subpopulations. EPC cells hold great promise in the field and have been shown to enhance proliferative ability, survival rate and angiogenic potential. Stem cell-derived EC also represent a viable alternative to directly isolating endothelial cells and its precursors, however issues including ethical concerns, source availability and tumourgenicity limit their application. Other cell types co-cultured with endothelial cells have also been shown to play both direct and indirect roles in the development and maturation of capillary networks.
The selection of appropriate scaffolds is also an important consideration. ECs require a 3D environment, with adhesion and degradation sites, in order to form functional tube structures with a lumen. EC capillary formation is also strongly associated with decreasing hydrogel stiffness. Modifying cell ratios can help prevent one cell type taking over the construct and/or push a co-culture system towards a particular desired cell trait. When optimizing cell culture media, researchers need to take into account the factors released by both cell types as it changes the dynamics of the culture. Supplements that previously supported a cell type in monoculture may not be required or may even have a detrimental effect on cells in co-culture. Finally, specialized seeding techniques and dynamic bioreactors can be used to overcome barriers in co-culture systems, but the optimal strategy will depend on the desired outcome.
Balancing all these conditions can be difficult, and with increasing number of novel biomaterials, cell isolation strategies, media formulations, seeding techniques and bioreactor systems being developed, the variety of options available to researchers is only set to continue. However, it is important for researchers to be able to identify parameters, understand the interrelationship between variables and appreciate the knock-on effect that changing of different conditions can have on a co-culture system, in order to help them appropriately design their experiments and achieve the desired research outcomes.
K Khosrotehrani was supported by the NHMRC Australia (project grant 1023368 and fellowship 1023371).
Authors’ original submitted files for images
Below are the links to the authors’ original submitted files for images.
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