Microtissue size and hypoxia in HTS with 3D cultures DRUDIS 1002 1–8 Fe a tu re s � P E R S P E C T IV E feature Microtissue size and hypoxia in HTS with 3D cultures Amish Asthana and William S. Kisaalita*, williamk@engr.uga.edu The three microenvironmental factors that characterize 3D cultures include: first, chemical and/or biochemical composition, second, spatial and temporal dimensions, and third, force and/or substrate physical properties. Although these factors have been studied individually, their interdependence and synergistic interactions have not been well appreciated. We make this case by illustrating how microtissue size (spatial) and hypoxia (chemical) can be used in the formation of physiologically more relevant constructs (or not) for cell-based high-throughput screening (HTS) in drug discovery. We further show how transcriptomic and/or proteomic results from heterogeneously sized microtissues and scaffold architectures that deliberately control hypoxia can misrepresent and represent in vivo conditions, respectively. We offer guidance, depending on HTS objectives, for rational 3D culture platform choice for better emulation of in vivo conditions. Drug Discovery Today �Volume 00, Number 00 �April 2012 PERSPECTIVE Traditionally, the meaning of three-dimension- ality in cell culture has been simply associated with providing a 3D spatial microenvironment. In our recent work, the meaning has been extended to providing the total microenviron- ment that supports the formation of microtissue that exhibit ‘complex’ physiological relevance (CPR) or better emulation of the in vivo micro- tissue functionality in a manner not possible in 2D cultures [1]. The literature has provided guidance that lead to three main categories or microenvironment factors (MEFs) or ‘three- dimensions’ of: first, chemical or biochemical composition, second, spatial (geometric 3D) and temporal dimensions, and third, force and sub- strate physical properties [1–4]. Although much attention has been paid to biochemical factors, such as integrin adhesion and presence of RDG peptides (integrin recognition site) in 3D cultures Please cite this article in press as: Asthana, A. Microtissue s 1359-6446/06/$ - see front matter � 2012 Published by Elsevier Ltd. d and biophysical cues, such as loading and unloading of collagen matrices, interactions among the factors have not been well studied. The effect of the aforementioned microen- vironmental factors is not exclusive, but they act synergistically to propel the cells towards a specific outcome. For instance, although regu- lating the size of the microtissue might seem trivial, it might indirectly have major implications on the functional response of the cells. If the tissue formed is too small, it might lack the physiologically relevant complexity and might not emulate the complex functionality present in vivo. Conversely, if the tissue is large (spatial factor), the oxygen diffusion (biochemical factor) limitation might lead to a necrotic core, reducing the viability and influencing the phenotypic outcome. It is known that oxygen can diffuse across 100–200 mm of tissue thickness and it is ize and hypoxia in HTS with 3D cultures, Drug Discov Today oi:10.1016/j.drudis.2012.03.004 generally desirable to maintain the optimal aggregate size approximately 250 mm to pre- vent hypoxia [2]. However, this size should not be treated as a gold standard, as the optimal size might depend on the application. In the field of regenerative medicine, circumventing hypoxia to produce larger tissues with higher viability for implantation in vivo is relentlessly pursued, however, the field of drug discovery might benefit from incorporating hypoxia in platform design. After all, hypoxia is a physiologically relevant phenomenon and is important for many in vivo processes, such as development and tumor progression. Indeed hypoxia has been widely implicated as the initiator of angiogenesis in avascular tumors, vasculogenesis during embryonic development and regulator of terminal differentiation [5]. As such, it is impor- tant to regulate the size of the tissue in 3D (2012), doi:10.1016/j.drudis.2012.03.004 www.drugdiscoverytoday.com 1 http://dx.doi.org/10.1016/j.drudis.2012.03.004 http://dx.doi.org/10.1016/j.drudis.2012.03.004 PERSPECTIVE Drug Discovery Today �Volume 00, Number 00 �April 2012 DRUDIS 1002 1–8 TABLE 1 Microtissue size control in the commercially available 3D platforms Company Trade name Type and material Cell aggregate/ scaffold pore size 3DBiomatrix Perfecta3D plates Hanging drops NA Perfecta3D scaffolds Hydrogel NA InSphero GravityPlus plates Hanging drops NA BD Matrigel Laminin, Collagen NA Glycosan Biosystems Extracel Hyaluronic acid and Collagen NA GlobalCellSollutions/Hamilton GEM Magnetic alginate microcarrier NA Trevigen Cultrex 3D Matrix BME, Laminin, Collagen NA Sigma HydroMatrix Synthetic Peptide Hydrogel NA MaxGel Human ECM NA QGel MT 3D Matrix Hydrogel NA Kollodis BioSciences MAPTrix HyGel Chemically defined Hydrogel NA Synthecon Inc. BIOFELT PGA, PLLA, PLGA, custom NR Biomerix 3D Scaffold Polycarbonate polyurethane-urea 100–250 Invitrogen Geltrex Laminin, Collagen NA AlgiMatrix Alginate 40–300 ZellWerk Sponceram Ceramic NA amsbio alvetex Polystyrene NR 3DM Inc. PuraMatrix Peptide 50–400 Corning UltraWeb Polyamide 300–500 3DBiotek 3D Insert PCL Polycaprolactone 300; 500 3D Insert PS Polystyrene 200; 400 3D Insert PLGA Poly(DL-lactide-co-glycolide) 500 b-TCP Disc b-Tricalcium phosphate NR MicroTissues Inc. 3D Petri Dish Agarose Multiple Abbreviations: NA, not applicable; NR, not reported. Fe a tu re s � P E R S P E C T IV E culture to include or eliminate the effects of hypoxia depending on the physiological phe- nomenon of interest. To illustrate further, in anticancer drug discovery, test compounds might be scored ‘hits’ with early stage tumor progression models, however, these compounds might be incompetent if tested with a micro- tissue tumor model having a larger size, owing to the drug resistance associated with hypoxic tumors. To make the case for the importance of microtissue size, we present many 2D and/or 3D comparative transcriptomic and proteomic stu- dies where the effect of the culture platform studied, might have been confounded by the heterogeneous microtissue sizes and thus the effect of hypoxia on gene expression was affected. Taken together, we stress the impor- tance of inclusion of hypoxic conditions in constructs through size regulation for certain applications. Such a realization is important in rational design and/or choice of a 3D platform, where the need for strict control of microtissues is balanced against the need for flexibility to alter microtissue size to better emulate the in vivo conditions. Please cite this article in press as: Asthana, A. Microtissue s 2 www.drugdiscoverytoday.com Spatial constraints in 3D platforms Most of the 3D platforms that are commercially available (Table 1) can be broadly classified into three categories based on the spatial constraints that they impose. First, Hydrogel-forming (algi- nate, agarose, chitosan, fibrin, hyaluronan and collagen to name a few) that cannot exert control on the size of the microtissues giving rise to heterogeneous microtissues (Type I; Fig. 1a). In such constructs, the microtissues range from being just a cluster of few cells to larger tissues that are above the crucial size for oxygen dif- fusion and might provide an adulterated response as discussed in the previous section. The sizes of the tissues depend on the seeding density and the proliferation rate of the cells and because the scaffold material is pliable there is no physical constraint on the size of the aggregates formed. Recently, a gradient depressurization strategy has been used to control the porosity and pore diameter in chit- osan hydrogels. However whether this technol- ogy is successful with hydrogels made of other substrates remains to be seen [6]. Scaffold-free spheroid cultures also fall into the same category ize and hypoxia in HTS with 3D cultures, Drug Discov Today because their size also depends on the afore- mentioned factors rather than physical restric- tions. The sizes of the aggregates growing in rotary wall vessels (RWV) or continuous stirred- tank reactors (CSTR) can be regulated by physical factors, such as revolutions per minute (rpm) and fluid shear stress, however; this control is acti- vated only when the spheroids grow above a crucial size. Moreover, the larger tissues are broken down into random smaller sizes giving rise to heterogeneously sized microtissues. Also, smaller tissues present in the culture, ranging from single cells to just below the threshold limit are not affected by this mechanism. Second, Synthetic microporous scaffolds made of stiff materials (e.g. polymers, such as polystyrene) that put a physical restraint on the size of the microtissues but the range of the pore size is large, again resulting in a variable sized popu- lation (Type II; Fig. 1b). However, the extent of variation is lower than Type I cases and the variability is defined as the range of pore sizes is known to the user. Third, Polymeric scaffolds that have a defined geometry and homogeneous pore sizes and provide a strict spatial control on (2012), doi:10.1016/j.drudis.2012.03.004 http://dx.doi.org/10.1016/j.drudis.2012.03.004 Drug Discovery Today �Volume 00, Number 00 �April 2012 PERSPECTIVE DRUDIS 1002 1–8 a b c Drug Discovery Today FIGURE 1 Three categories of 3D platforms. (a) Hydrogel (AlgiMatrix) with heterogeneous (C3A human hepatocytes) microtissues stained with live/dead dye kit. Dead cells (stained red) are visible at the center of large aggregates [51]. (b) Polystyrene scaffold that imposes a size constraint but offers a wide range of pore sizes. The scaffold was fabricated following procedures described in [52]. (c) SU-8 (photoresist material) microwell scaffold that provides a uniform pore size. The scaffold was fabricated following procedures described in [53]. Scale bars = 100 mm. Fe a tu re s � P E R S P E C T IV E the size of the microtissues (Type III; Fig. 1c). If seeded at the optimal cell density, such scaffolds produce a population of equally sized aggre- gates; however they might lack flexibility and can be application specific. For instance, a Please cite this article in press as: Asthana, A. Microtissue s scaffold having a defined pore size of 200 mm might not be suited for studying or developing a drug against a late stage cancer, where the tumor is hypoxic and adapting for angiogenesis. Conversely, while developing tissue models for ize and hypoxia in HTS with 3D cultures, Drug Discov Today drug testing by differentiating stem cells, a larger pore size (500 mm) might produce microtissues with hypoxic cores and this might influence the differentiation capacity of the cells, giving rise to a heterotypic model. The impact of hypoxia in both these scenarios is discussed below. Owing to such variability, the response generated due to a specific treatment by cells growing on different 3D platforms might be different and too difficult to compare. One must consider that variability in tissue size might manifest itself in the form of hypoxia or other unknown forms and perturb gene expression leading to an adulter- ated response to the administered treatment. Relationship between microtissue size and gene expression As discussed above, if the size of the microtissue grows beyond the threshold for oxygen diffu- sion, the cells in the core of the aggregate become hypoxic. Hypoxia can manifest itself in the form of gene expression perturbation because it regulates the expression of a wide variety of genes associated with oxygen trans- port and iron metabolism, glycolysis and glucose uptake, angiogenesis, extracellular matrix (ECM) and coagulation systems, drug resistance, pH regulation, transcription and growth factors and cytokines (Table 3) [7,8]. The relationship between microtissue size and gene expression was established by Kelm et al. [9] where it was shown that larger myocardial spheroids (230 � 11 mm in diameter) produced high levels of vascular endothelial growth factor (VEGF; a marker of hypoxia) while it was absent in smaller spheroids (130 � 11 mm in diameter) although both the sizes showed CPR (synchronized beating frequencies). This relationship is further sub- stantiated by a transcriptomic study [10] showing that neural progenitor (NP) cells growing as neurospheres (larger size) had a higher number of upregulated genes than those growing in 3D microporous scaffolds (controlled smaller size) when compared with 2D cultures. It was sug- gested that this might be owing to hypoxia associated with the larger size of neurospheres as was evident by the upregulation of macrophage inflammatory protein-2 (MIP-2) gene, which is induced by hypoxic conditions [11]. Several transcriptomic and proteomic studies that have compared gene expression variations between a variety of cells grown in 2D and 3D formats are listed in Table 2. These differential gene expression events have generally been solely attributed to the transition from 2D to a 3D platform, but hypoxia associated with larger tissue size might also be responsible for these gene perturbations as several of them might be (2012), doi:10.1016/j.drudis.2012.03.004 www.drugdiscoverytoday.com 3 http://dx.doi.org/10.1016/j.drudis.2012.03.004 PERSPECTIVE Drug Discovery Today �Volume 00, Number 00 �April 2012 DRUDIS 1002 1–8 TABLE 2 Hypoxia controlled genes/proteins found upregulated in 3D/2D comparative studies. Cell line 3D Scaffold Size (mm) Genesa Refs Fibroblasts Collagen matrix ND IL-6 [41] IMR-90 Collagen–GAG matrix 80–100 HIG2, IL-8, CXCL2, VEGF, FTH1, FTL [42] MG-63, SaOS-2 Si-HPMC polymer hydrogel ND IL-6 [43] NA8 Spheroids on pHEMA plates ND IL-8, CXCL2, Angiopoetin like4, CA-9, LOX, ADM, HIG2, BNip3, IGFBP3, Jun, ITGA2 [44] R1 Cytomatrix RW-spinner culture 4150 Jun, IGF-2 [45] L1236 RADA-oligopeptide matrix ND CCL5, TNF [46] PDAC pECM ND IL-6, IL-8 [47] OSCC3, U87 MDA-MB231 PLG, RGD alginate, Matrigel ND IL-8, VEGF [48] HFSF, CRL-2088 HES HAL MRC-5 Spheroids on Agarose plates ND COX-2, CCL3,CCL5, CXCL8 CXCL8 CXCL8 CXCL8 [49] BMSC Spheroids on Agarose plates 400 CXCL12 [50] aBold italic indicates protein results, other results are only transcriptomic. Abbreviations: IL, interleukin; CXCL2, Macrophage inflammatory protein 2; FTH1, Ferritin Heavy subunit; FTL, Ferritin Light subunit; HIG2, hypoxia-inducible gene 2 protein; ND, Not Defined. Fe a tu re s � P E R S P E C T IV E missing in 3D cultures of smaller sizes [9,10]. The genes listed in Table 2 were found to be upre- gulated in 3D cultures when compared to 2D but they were also induced by hypoxia (Table 3) suggesting that gene expression due to hypoxic conditions might have been involved in these studies. Thus, to assess the genes whose expression is significantly altered specifically owing to 3D culture conditions, the size of the microtissue should be controlled; else the genes influenced by hypoxia might augment the total number of differentially expressed genes and mask the ones that are really of interest. Physiological relevance of hypoxia – need for inclusion in construct design Hypoxia can be a physiologically relevant phe- nomenon because it is a major characteristic of 3D microenvironments both in vivo and in vitro. Oxygen concentration in 3D tissues depends on the balance between oxygen delivery and con- sumption. In vivo, this balance is tightly regu- lated by evenly distributed capillary networks but in vitro homotypic 3D microtissues lack vasculature and therefore develop a hypoxic core as their size increases. This might lead to cells, producing chemical signals (cytokines) and programming themselves for developmental, adaptive or neoplastic angiogenesis depending upon their type (stem, committed or malignant, respectively). This is similar to the response generated by in vivo hypoxic tissues where balanced signaling cascades lead to vascular remodeling and angioadaptation until the tissue oxygen concentration is back within its normal Please cite this article in press as: Asthana, A. Microtissue s 4 www.drugdiscoverytoday.com range [12]. The central connection between physiological hypoxia and the cellular response is mediated by hypoxia inducible transcription factors (HIFs). Under hypoxic conditions, HIF-1a and HIF-1b are translocated to the nucleus [13] where they dimerize and bind to target gene motifs called hypoxia responsive elements (HREs) to alter gene expression [14]. In vivo, hypoxia is generally associated with the tumor microenvironment where it is responsible for angiogenesis, drug resistance and increased metastatic potential of the malignant cells and also with development where it regulates vas- culogenesis, stem cell renewal and terminal differentiation. As such, hypoxic conditions need to be included in the 3D scaffold design, where applicable, to better mimic these conditions in vitro. This can be done in a physiologically relevant manner by strictly regulating the size of the microtissue. Hypoxia and the tumor microenvironment Neoplastic angiogenesis is an essential process in tumor progression and the initiation of metastasis [15]. The phenomenon of angiogen- esis comprises a series of linked and sequential steps that finally leads to the development of a neovascular blood supply to the tumorous tissue [16]. Angiogenic growth factors secreted by infiltrating immune cells, adjacent stroma and tumor cells themselves bind to specific receptors on endothelial cells which leads to endothelial cell proliferation, migration and invasion, even- tually culminating in capillary formation. ize and hypoxia in HTS with 3D cultures, Drug Discov Today Angiogenesis regulation by hypoxia is an important homeostatic mechanism that links vascular oxygen supply to metabolic demand. Lately, molecular characterization of angiogenic pathways, establishment of HIFs as their key transcriptional regulators and the identification of hydoxylases that regulate HIF corresponding to the oxygen availability have provided novel insights into this process [17]. Hypoxia in the tumor microenvironment is of specific impor- tance in drug discovery and development because optimal oxygenation is a primary requisite for many chemotherapeutic drugs, such as alkylating agents (melphalan), antibio- tics (bleomycin) and podophyllotoxins (etopo- side) to act at their maximum efficiency [7]. Most alkylating agents act by transferring alkyl groups to DNA during cell division, following which the DNA strand breaks or cross-linking of the two strands occurs, preventing subsequent DNA synthesis [18,19]. Hypoxic conditions can lead to resistance against these drugs directly by increased production of nucleophilic sub- stances, such as glutathione, which might compete with the target DNA for alkylation, subsequently reducing the drug efficacy [20]. Another class of anticancer agents acts at spe- cific phases of the cell cycle. As hypoxia causes the cell cycle to slow down or lead to pre-S- phase arrest in extreme conditions [21], it can also indirectly affect the apoptotic potential of these agents. Also, hypoxia leads to changes in the genome that can confer growth advantage to cells with p53 mutations [22] or deficient in DNA mismatch repair [23], while inhibiting (2012), doi:10.1016/j.drudis.2012.03.004 http://dx.doi.org/10.1016/j.drudis.2012.03.004 Drug Discovery Today �Volume 00, Number 00 �April 2012 PERSPECTIVE DRUDIS 1002 1–8 TABLE 3 Genes induced by hypoxia Biological function Gene (abbreviation) Refs O2 transport and iron metabolism Erythropoietin (Epo) [7] Ferritin (FTH1, FTL) [7] Heme oxygenase-1 [7] Transferrin [7] Transferrin receptor (Tfr) [54] Ceruloplasmin [55] Angiogenesis Vascular endothelial growth factor (VEGF) [7,56] VEGF receptor-1 [7] Cyclooxygenase (COX)-2 [7] Leptin (LEP) [57] Endothelin-1, -2 [7] Fibroblast growth factor (FGF)-3 [7] Angiopoietin-4 (Ang-4) [58] Nitric oxide synthase (NOS) [7] Placental growth factor (PlGF) [7] Transforming growth factor (TGF)-a [59] TGF-b1 [7] TGF-b3 [7] Matrix metabolism and coagulation Metalloproteinases [7] Matrix metalloproteinase (MMP)-13 [7] Plasminogen activator inhibitor-1 [7] Urokinase receptor [7] Collagen prolyl hydroxylase [60] a-Integrin [7] Glycolysis and glucose metabolism Adenylate kinase-3 [8] Aldolase-A,C (ALDA,C) [8] Carbonic anhydrase-9 (CA-9) [61] Enolase-1 (ENO1) [8] Glucose transporter-1,3 (GLUT1,3) [7,62] Glyceraldehyde phosphate dehydrogenase (GAPDH) [8] Hexokinase 1,2 (HK1,2) [63] Lactate dehydrogenase-A (LDHA) [8] Pyruvate kinase M (PKM) [8] Phosphofructokinase L (PFKL) [8] Phosphoglycerate kinase 1 (PGK1) [8] 6-phosphofructo-2-kinase/gructose-2,6-bisphosphate-3 (PFKFB3) [64] Transcription factors Hypoxia-inducible factor (HIF)-1a [7] HIF-2a [7] Activator protein (AP-1) [7] Jun [7] Nuclear factor-kB (NF-kB) [7] Insulin-like growth factor (IGF) binding protein-1,-2, -3 [7] Cyclic AMP responsive-element-binding protein (CREB) [7] Drug resistance Multi-drug resistance (MDR1) [7] Apoptosis Bcl-2/adenovirus EIB 19kD-interacting protein 3 (BNip3) [65] Nip3-like protein X (NIX) [66] Growth factors and/or cytokines Insulin-like growth factor-2 (IGF-2) [7] Platelet-derived growth factor (PDGF) [7] Adrenomedullin (ADM) [8] Interleukin-6 (IL-6) [67] Interleukin-8 (IL-8; CXCL8) [67,68] Tumor Necrosis Factor a (TNFa) [67] Macrophage inflammatory protein 1-a (CCL3; MIP-1a) [69] Macrophage inflammatory protein 2 (MIP-2; CXCL2) [11] Stromal cell-derived factor 1 (SDF-1; CXCL12) [70] Chemokine (C–C motif) ligand (CCL5; RANTES) [71] Fe a tu re s � P E R S P E C T IV E apoptosis, which also contributes to their therapeutic resistance. Screening of such drug compounds on the aforementioned Type I and II 3D platforms, that produce microtissues of variable sizes, can lead to Please cite this article in press as: Asthana, A. Microtissue s skewed responses because the compounds might be selectively effective against a certain popula- tion of cells. For such compounds, Type III plat- forms seem to be the more obvious choice, but the size cut-off that is meant for this particular ize and hypoxia in HTS with 3D cultures, Drug Discov Today application must be carefully chosen. Also, the difference between hypoxic cancer cells and normal cells provides a novel target upon which cytotoxic drugs can be designed. These drug designing strategies include: targeting the HIF-1 (2012), doi:10.1016/j.drudis.2012.03.004 www.drugdiscoverytoday.com 5 http://dx.doi.org/10.1016/j.drudis.2012.03.004 PERSPECTIVE Drug Discovery Today �Volume 00, Number 00 �April 2012 DRUDIS 1002 1–8 Fe a tu re s � P E R S P E C T IV E transcription factor (thioredoxin-1 inhibitors – pleurotin, PX-12; [24]), hypoxia-selective gene therapy, pro-drugs activated by hypoxia (AQ4N, NLCQ-1) and the use of recombinant obligate anaerobic bacteria (non-pathogenic clostridia) [25]. As such, these novel strategies require a consistent population of hypoxic microtissues to be tested upon. One might argue that culturing cells under reduced oxygen pressure or adding a hypoxia-mimetic agent, such as desferrioxamine mesylate (DFX; iron chelator) to the media can produce hypoxic growth conditions but this might not recreate the natural oxygen and nutrient gradients that are associated with the tumor microenvironment and a 3D culture of larger hypoxic microtissues might be a better emulation of the in vivo situation, rendering the platforms that do not provide control on size or produce microtissues below the required size, less desirable for such applications. Recently, a report published by Rohwer and Cramer [26] listed several studies where hypoxic conditions and HIFs have been directly implicated in chemothera- peutic resistance leading to inefficacy of drugs along with the resistance phenotypes and the molecular mechanisms underlying the resistance. Had the potency of these drugs been validated on 3D microtissues of a particular size that incorpo- rated a hypoxic core, it is plausible that they might have been rejected during the initial phase of discovery and this would have saved considerable resources. Hypoxia and stem cell niches The availability of reliable cell types is the pri- mary concern in the successful development of therapeutics against cellular targets in the drug discovery process. These cells are typically obtained from primary tissue, immortalized tumor cells or genetically transformed cell lines. Compared with primary and immortalized cells, stem cells are more advantageous as they are genetically normal and can be maintained in culture for a longer time, increasing their applicability in the screening process. However, to fully exploit their potential, successful and consistent protocols need to be developed for their renewal and maintenance and also differ- entiation into committed lineages. Recently, many studies have shown that 3D cultures consistently outperform 2D monolayer cultures in terms of promoting stem cell growth, differ- entiation and development of complex physio- logically relevant structures and functionality. When compared with 2D, embryonic stem cells (ESC) grown in 3D cultures, differentiated into hepatocytes that had closer resemblance to their in vivo counterparts in terms of morphology, Please cite this article in press as: Asthana, A. Microtissue s 6 www.drugdiscoverytoday.com gene expression and biological behavior [27,28]. Similar results have been obtained with 3D cultures of stem cells differentiated into cells of neural [29–31], epithelial [29], endothelial [29,30], chondrogenic [30,32], and hematopoie- tic [33] lineages. Moreover, 3D cultures have also been shown to sustain long-term self-renewal of human ESCs, maintaining them in undifferen- tiated state, preserving their normal karyotype, and conserving their differentiation capacity as indicated by embryoid body formation [34]. Considering an increasing shift from the usage of traditional cultures towards 3D platforms for maintaining or differentiating stem cells to generate complex models of particular tissue types for drug testing, the hypoxic conditions associated with the 3D microenvironment should be taken into account as oxygen tension has been known to influence stem cell quies- cence, proliferation and differentiation both in vivo and in vitro [35]. It has been shown that hematopoietic stem cells (HSCs) are more likely to reside in the low oxygen areas of the marrow, away from blood vessels and hypoxia appears to regulate hematopoiesis in the bone marrow by influencing the survival, metabolism and cell cycle of HSC and also provides protection against oxidative stress [36]. Also, the differen- tiation capability of bone marrow-derived mesenchymal stem cells (MSCs) is decreased in hypoxic culture conditions along with an increase in Oct-4 expression and telomerase activity, further substantiating the notion that low oxygen tension promotes an undifferen- tiated state of these stem cells [37]. Similarly, hypoxia also promotes survival, proliferation and maintenance of an undifferentiated state in neural crest stem cells and NSCs [38]. Hypoxic (physiologically normoxic) culture conditions also seem to maintain full pluripotency and enhance embryoid body formation of human ESCs [39] whereas normal environmental oxygen levels lead to a significant decrease in the expression of stem cell markers, SOX2, Nanog and Oct-4 [40]. Furthermore, silencing of HIF-2a and HIF3-a, but not of HIF-1a, also led to a substantial reduction in the expression of the aforementioned pluripotency markers [40] further implicating a role of hypoxia in the maintenance of pluripotency and stemness. Recently, HIF-2a has been shown to bind to the Oct-4 promoter and induce its expression and transcriptional activity [5], thus hypoxia can contribute to generation of induced pluripotent stem cells (iPSC). As hypoxia has such an important role in determining stem cell fate, it is essential to regulate the size of the microtissue in 3D to either maintain the differentiation capacity ize and hypoxia in HTS with 3D cultures, Drug Discov Today of the cells or induce differentiation towards a particular lineage. Therefore a platform that does not maintain a homogenous population of equally sized microtissues might have a mixed heterotypic population of cells, some differen- tiated while others maintaining their differentia- tion capability, giving rise to differences in drug response. A mixed population is a physiologically relevant condition; however each microtissue should have the same distribution of each type of cells to generate a valid drug response. If the size of the aggregates is different, the smaller ones might have a higher proportion of differentiated cells whereas the larger ones might be replete with stem cells as their cores become hypoxic and maintain the stemness of the cells. Concluding remarks The tale of the spatial and the biochemical microenvironmental factors in 3D cell culture is that they are not mutually independent, because an increase in size leads to a depletion of oxygen and creates hypoxic growth conditions. Most of the commercially available 3D growth platforms either do not regulate the size of the microtissue or restrict it to prevent hypoxia. However, hypoxia is a physiologically relevant phenomenon encountered in avascular tumors and stem cell niches and affects the gene expression profiles of the cells. As such, it should be a key factor in rational design or selection of 3D HTS platforms for preclinical drug discovery. For example, to test the cytotoxic potential of chemotherapeutic agents, it might be more desirable to use hypoxic rather than smaller normoxic microtissues that might be more susceptible to their apoptotic actions and produce false positives. Similarly, for generation of tissue models from stem cells for drug screening, a size cutoff should be chosen according to the state of cells (renewal or differ- entiation) required for the application. Finally, in future transcriptomic and/or proteomic compar- ison studies, a marker for hypoxic conditions, such as lysyl oxidase (LOX) should be included or a fluorescent hypoxyprobe, such as Oxylite (Oxford Optonics; http://www.oxford-optronix.com/) should be used to determine whether the cells are hypoxic. Also, a subset of genes (Table 3) dedi- cated to hypoxia can be established and its hierarchy or the rank assigned to it by clustering programs might suggest the degree to which hypoxia might be responsible for results from differential gene expression studies. References 1 Kisaalita, W.S. (2010) 3D Cell-based Biosensors in Drug Discovery Programs: Microtissue Engineering for High Throughput Screening. CRC Press, Taylor & Francis Group (2012), doi:10.1016/j.drudis.2012.03.004 http://www.oxford-optronix.com/ http://dx.doi.org/10.1016/j.drudis.2012.03.004 Drug Discovery Today �Volume 00, Number 00 �April 2012 PERSPECTIVE DRUDIS 1002 1–8 Fe a tu re s � P E R S P E C T IV E 2 Griffith, L.G. and Swartz, M.A. (2006) Capturing complex 3D tissue physiology in vitro. Nat. Rev. Mol. Cell Biol. 7, 211–224 3 Green, J.A. and Yamada, K.M. (2007) Three-dimensional microenvironments modelate fibroblast signaling responses. Adv. Drug Deliv. Rev. 59, 1289–1293 4 Lai, Y. et al. 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Chem. 276, 43407–43412 (2012), doi:10.1016/j.drudis.2012.03.004 www.drugdiscoverytoday.com 7 http://dx.doi.org/10.1016/j.drudis.2012.03.004 PERSPECTIVE Drug Discovery Today �Volume 00, Number 00 �April 2012 DRUDIS 1002 1–8 Fe a tu re s � P E R S P E C T IV E 64 Minchenko, A. et al. (2002) Hypoxia-inducible factor-1- mediated expression of the 6-phospho-fructo-2- kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) gene. Its possible role in the Warburg effect. J. Biol. Chem. 277, 6183–6187 65 Carrero, P. et al. (2000) Redox-regulated recruitment of the transcriptional coactivators CREB-binding protein and SRC-1 to hypoxia-inducible factor 1alpha. Mol. Cell. Biol. 20, 402–415 66 Bruick, R.K. (2000) Expression of the gene encoding the proapoptotic Nip3 protein is induced by hypoxia. Proc. Natl. Acad. Sci. U.S.A. 97, 9082–9087 Please cite this article in press as: Asthana, A. Microtissue s 8 www.drugdiscoverytoday.com 67 Jeong, H.J. et al. (2003) Expression of proinflammatory cytokines via HIF-1a and NF-kB activation on desferrioxamine-stimulated HMC-1 cells. Biochem. Biophys. Res. Commun. 306, 805–811 68 Karakurum, M. et al. (1994) Hypoxic induction of interleukin-8 gene expression in human endothelial cells. J. Clin. Invest. 93, 1564–1570 69 Bosco, M.C. et al. (2004) Hypoxia selectively inhibits monocyte chemoattractant protein-1 production by macrophages. J. Immunol. 172, 1681–1690 70 Hitchon, C. et al. (2002) Hypoxia-induced production of stromal cell-derived factor 1 (CXCL12) and vascular ize and hypoxia in HTS with 3D cultures, Drug Discov Today endothelial growth factor by synovial fibroblasts. Arthritis Rheum. 46, 2587–2597 71 Skurk, T. et al. (2009) Expression and secretion of RANTES (CCL5) in human adipocytes in response to immunological stimuli and hypoxia. Horm. Metab. Res. 41, 183–189 Amish Asthana, William S. Kisaalita Cellular Bioengineering Laboratory, Driftmier Engineering Center, University of Georgia, Athens, GA 30602, United States (2012), doi:10.1016/j.drudis.2012.03.004 http://dx.doi.org/10.1016/j.drudis.2012.03.004 Microtissue size and hypoxia in HTS with 3D cultures Spatial constraints in 3D platforms Relationship between microtissue size and gene expression Physiological relevance of hypoxia - need for inclusion in construct design Hypoxia and the tumor microenvironment Hypoxia and stem cell niches Concluding remarks References