Functional scaffold-free 3-D cardiac microtissues: a novel model for the investigation of heart cells

To bridge the gap between two-dimensional cell culture and tissue, various three-dimensional (3-D) cell culture approaches have been developed for the investigation of cardiac myocytes (CMs) and cardiac fibroblasts (CFs). However, several limitations still exist. This study was designed to develop a cardiac 3-D culture model with a scaffold-free technology that can easily and inexpensively generate large numbers of microtissues with cellular distribution and functional behavior similar to cardiac tissue. Using micromolded nonadhesive agarose hydrogels containing 822 concave recesses (800 μm deep × 400 μm wide), we demonstrated that neonatal rat ventricular CMs and CFs alone or in combination self-assembled into viable (Live/Dead stain) spherical-shaped microtissues. Importantly, when seeded simultaneously or sequentially, CMs and CFs self-sorted to be interspersed, reminiscent of their myocardial distribution, as shown by cell type-specific CellTracker or antibody labeling. Microelectrode recordings and optical mapping revealed characteristic triangular action potentials (APs) with a resting membrane potential of -66 ± 7 mV (n = 4) in spontaneously contracting CM microtissues. Under pacing, optically mapped AP duration at 90% repolarization and conduction velocity were 100 ± 30 ms and 18.0 ± 1.9 cm/s, respectively (n = 5 each). The presence of CFs led to a twofold AP prolongation in heterogenous microtissues (CM-to-CF ratio of 1:1). Importantly, Ba(2+)-sensitive inward rectifier K(+) currents and Ca(2+)-handling proteins, including sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a, were detected in CM-containing microtissues. Furthermore, cell type-specific adenoviral gene transfer was achieved, with no impact on microtissue formation or cell viability. In conclusion, we developed a novel scaffold-free cardiac 3-D culture model with several advancements for the investigation of CM and CF function and cross-regulation.     

Heterologous protein production capacity of mammalian cells cultivated as monolayers and microtissues

A precise understanding of processes managing heterologous protein production in vitro and in vivo is essential for the manufacture of sophisticated biopharmaceuticals as well as for future gene therapy and tissue engineering initiatives. Capitalizing on the gravity-enforced self-assembly of monodispersed cells into coherent (multicellular) microtissues we studied heterologous protein production of microtissues and monolayers derived from cell lines and primary cells engineered/transduced for (i) constitutive, (ii) proliferation-controlled, (iii) macrolide-, or (iv) gas-inducible expression of the human placental secreted alkaline phosphatase (SEAP) and of the Bacillus stearothermophilus-derived secreted alpha-amylase (SAMY). Specific productivity of cells assembled in microtissues was up to 20-fold higher than isogenic monolayer cultures. Diffusion across microtissues could be further increased by HUVEC-mediated vascularization. As well as higher specific protein productivities, microtissues were also more efficient than monolayer cultures in assembling transgenic lentiviral particles. Our results showed that mammalian cells embedded in a tissue-like three-dimensional (3D) microenvironment exhibit increased production capacity. This observation should be considered for gene therapy and tissue engineering scenarios as well as for biopharmaceutical manufacturing.     

VEGF profiling and angiogenesis in human microtissues

Owing to its dual impact on tissue engineering (neovascularization of tissue implants) and cancer treatment (prevention of tumor-induced vascularization), management and elucidation of vascularization phenomena remain clinical priorities. Using a variety of primary human cells and (neoplastic) cell lines assembled in microtissues by gravity-enforced self-aggregation in hanging drops we (i) studied size and age-dependent VEGF production of microtissues in comparison to isogenic monolayer cultures, (ii) characterized the self-organization and VEGF-production potential of mixed-cell spheroids, (iii) analyzed VEGF-dependent capillary formation of human umbilical vein endothelial cells (HUVECs) cells coated onto several human primary cell spheroids, and (iv) profiled endostatin action on vascularization in human microtissues. Precise understanding of vascularization in human microtissues may foster advances in clinical tissue implant engineering, tumor treatment, as well as drug discovery and drug-function analysis.     

A scaffoldless technique for self-generation of three-dimensional keratinospheroids on liquid crystal surfaces

We describe a new scaffold-free three-dimensional (3D) cell culture model using cholesteryl ester based lyotropic liquid crystal (LC) substrates. Keratinocytes were deposited randomly on the LC surface where they self-assembled into 3D microtissues or keratinospheroids. The cell density required to form spheroids was optimized. We investigated cell viability using dead/live cell assays. The adhesion characteristics of cells within the microtissues were determined using histological sectioning and immunofluorescence staining. Fourier transform infrared spectroscopy (FTIR) was used to characterize the biochemistry of the keratinospheroids. We found that both cells and microtissues could migrate on the LC surface. The viability study indicated approximately 80% viability of cells in the microtissues up to 20 days of culture. Strong intercellular adhesion was observed in the stratification of the multi-layered microspheroids using field emission-scanning electron microscopy (FE-SEM) and histochemical staining. The cytoskeleton and vinculins of the cells in the microtissues were expressed diffusely, but the microtissues were enriched with lipids and nucleic acids, which indicates close resemblance to the conditions in vivo. The basic 3D culture model based on LC may be used for cell and microtissue migration studies in response to cytochemical treatment.     

Controlling cell position in complex heterotypic 3D microtissues by tissue fusion

Tissue fusion and cell sorting are processes fundamental to developmental biology with applications in tissue engineering. We have designed a fusion assay to investigate the factors governing the fusion of microtissues and the cell sorting that occurs after fusion. Normal human fibroblast (NHF) spheroids were self‐assembled and cultured for 1, 4, or 7 days, then combined in trough shaped recesses. Over a 24‐h period the spheroids fused to become a rod shaped microtissue and the kinetics and extent of fusion could be quantified by assessing rod contraction. By varying the amount of spheroid culture time prior to fusion (1–7 days), the rate of fusion, the coherence of the building units (as measured by fusion angle) and the steady state length of the structure could be easily controlled. Longer pre‐culture times for the spheroids resulted in slower fusion, less coherence and increased length of rod microtissues. The fusion kinetics and steady length of rods formed by smaller versus larger spheroids (～100 vs. 300 µm diameter) were indistinguishable, even though smaller spheroids had twice the surface area and greater numbers of contacts between units. Both small and large spheroids were strongly influenced by spheroid pre‐culture time. Pre‐culture time could also be used to control cell sorting and cell position when combinations of NHFs and H35s, a rat hepatocyte cell line, were fused to form heterotypic microtissues. Control of fusion and cell position are important parameters for creating functional heterotypic microtissues as well as the use of microtissues as building units to create larger tissue structures. Biotechnol. Bioeng. 2009;102: 1231–1241. © 2008 Wiley Periodicals, Inc.     

Three-Dimensional Scaffold-Free Fusion Culture: the Way to Enhanced Chondrogenesis of in vitro Propagated Human Articular Chondrocytes

Cartilage regeneration based on isolated and culture-expanded chondrocytes has been studied in various in vitro models, but the quality varies with respect to the morphology and the physiology of the synthesized tissues. The aim of our study was to promote in vitro chondrogenesis of human articular chondrocytes using a novel three-dimensional (3-D) cultivation system in combination with the chondrogenic differentiation factors transforming growth factor beta 2 (TGF-β2) and L-ascorbic acid. Articular chondrocytes isolated from six elderly patients were expanded in monolayer culture. A single-cell suspension of the dedifferentiated chondrocytes was then added to agar-coated dishes without using any scaffold material, in the presence, or absence of TGF-β2 and/or L-ascorbic acid. Three-dimensional cartilage-like constructs, called single spheroids, and microtissues consisting of several spheroids fused together, named as fusions, were formed. Generated tissues were mainly characterized using histological and immunohistochemical techniques. The morphology of the in vitro tissues shared some similarities to native hyaline cartilage in regard to differentiated S100-positive chondrocytes within a cartilaginous matrix, with strong collagen type II expression and increased synthesis of proteoglycans. Finally, our innovative scaffold-free fusion culture technique supported enhanced chondrogenesis of human articular chondrocytes in vitro. These 3-D hyaline cartilage-like microtissues will be useful for in vitro studies of cartilage differentiation and regeneration, enabling optimization of functional tissue engineering and possibly contributing to the development of new approaches to treat traumatic cartilage defects or osteoarthritis.Key words: in vitro cartilage, 3-D cell culture, fusion culture technique, tissue engineering, cell differentiation, extracellular matrix, immunohistochemistry     

Self-assembly of sensory neurons into ganglia-like microtissues

Unraveling intra- and inter-cellular signaling networks managing cell-fate control, coordinating complex differentiation regulatory circuits and shaping tissues and organs in living systems remain major challenges in the post-genomic era. Resting on the laurels of past-century monolayer culture technologies, the cell culture community has only recently begun to appreciate the potential of three-dimensional mammalian cell culture systems to reveal the full scope of mechanisms orchestrating the tissue-like cell quorum in space and time. Capitalizing on gravity-enforced self-assembly of monodispersed primary embryonic mouse cells in hanging drops, we designed and characterized a three-dimensional cell culture model for ganglion-like structures. Within 24h, a mixture of mouse embryonic fibroblasts (MEF) and cells, derived from the dorsal root ganglion (DRG) (sensory neurons and Schwann cells) grown in hanging drops, assembled to coherent spherical microtissues characterized by a MEF feeder core and a peripheral layer of DRG-derived cells. In a time-dependent manner, sensory neurons formed a polar ganglion-like cap structure, which coordinated guided axonal outgrowth and innervation of the distal pole of the MEF feeder spheroid. Schwann cells, present in embryonic DRG isolates, tended to align along axonal structures and myelinate them in an in vivo-like manner. Whenever cultivation exceeded 10 days, DRG:MEF-based microtissues disintegrated due to an as yet unknown mechanism. Using a transgenic MEF feeder spheroid, engineered for gaseous acetaldehyde-inducible interferon-beta (ifn-beta) production by cotransduction of retro-/ lenti-viral particles, a short 6-h ifn-beta induction was sufficient to rescue the integrity of DRG:MEF spheroids and enable long-term cultivation of these microtissues. In hanging drops, such microtissues fused to higher-order macrotissue-like structures, which may pave the way for sophisticated bottom-up tissue engineering strategies. DRG:MEF-based artificial micro- and macrotissue design demonstrated accurate key morphological aspects of ganglions and exemplified the potential of self-assembled scaffold-free multicellular micro-/macrotissues to provide new insight into organogenesis.     

Dissecting coronary angiogenesis: 3D co-culture of cardiomyocytes with endothelial or mesenchymal cells

In embryogenesis, coronary blood vessels are formed by vasculogenesis from epicardium-derived progenitors. Subsequently, growing or regenerating myocardium increases its vasculature by angiogenesis, forming new vessels from the pre-existing ones. Recently, cell therapies for myocardium ischemia that used different protocols have given promising results, using either extra-cardiac blood vessel cell progenitors or stimulating the cardiac angiogenesis. We have questioned whether cardiomyocytes could sustain both vasculogenesis and angiogenesis. We used a 3D culture model of tissue-like spheroids in co-cultures of cardiomyocytes supplemented either with endothelial cells or with bone marrow-derived mesenchymal stroma cells. Murine foetal cardiomyocytes introduced into non-adherent U-wells formed 3D contractile structures. They were coupled by gap junctions. Cardiomyocytes segregated inside the 3D structure into clumps separated by connective tissue septa, rich in fibronectin. Three vascular endothelial growth factor isoforms were produced (VEGF 120, 164 and 188). When co-cultured with human umbilical cord endothelial cells, vascular structures were produced in fibronectin-rich external layer and in radial septa, followed by angiogenic sprouting into the cardiomyocyte microtissue. Presence of vascular structures led to the maintenance of long-term survival and contractile capacity of cardiac microtissues. Conversely, bone marrow mesenchymal cells formed isolated cell aggregates, which progressively expressed the endothelial markers von Willebrand's antigen and CD31. They proceeded to typical vasculogenesis forming new blood vessels organised in radial pattern. Our results indicate that the in vitro 3D model of cardiomyocyte spheroids provides the two basic elements for formation of new blood vessels: fibronectin and VEGF. Within the myocardial environment, endothelial and mesenchymal cells can proceed to formation of new blood vessels either through angiogenesis or vasculogenesis, respectively.     

Differential Sensitivity of Epithelial Cells to Extracellular Matrix in Polarity Establishment

Establishment of apical-basal polarity is crucial for epithelial sheets that form a compartment in the body, which function to maintain the environment in the compartment. Effects of impaired polarization are easily observed in three-dimensional (3-D) culture systems rather than in two-dimensional (2-D) culture systems. Although the mechanisms for establishing the polarity are not completely understood, signals from the extracellular matrix (ECM) are considered to be essential for determining the basal side and eventually generating polarity in the epithelial cells. To elucidate the common features and differences in polarity establishment among various epithelial cells, we analyzed the formation of epithelial apical-basal polarity using three cell lines of different origin: MDCK II cells (dog renal tubules), EpH4 cells (mouse mammary gland), and R2/7 cells (human colon) expressing wild-type α-catenin (R2/7 α-Cate cells). These cells showed clear apical-basal polarity in 2-D cultures. In 3-D cultures, however, each cell line displayed different responses to the same ECM. In MDCK II cells, spheroids with a single lumen formed in both Matrigel and collagen gel. In R2/7 α-Cate cells, spheroids showed similar apical-basal polarity as that seen in MDCK II cells, but had multiple lumens. In EpH4 cells, the spheroids displayed an apical-basal polarity that was opposite to that seen in the other two cell types in both ECM gels, at least during the culture period. On the other hand, the three cell lines showed the same apical-basal polarity both in 2-D cultures and in 3-D cultures using the hanging drop method. The three lines also had similar cellular responses to ECM secreted by the cells themselves. Therefore, appropriate culture conditions should be carefully determined in advance when using various epithelial cells to analyze cell polarity or 3-D morphogenesis.     

Comparison of biophysical properties characterized for microtissues cultured using microencapsulation and liquid crystal based 3D cell culture techniques

Growing three dimensional (3D) cells is an emerging research in tissue engineering. Biophysical properties of the 3D cells regulate the cells growth, drug diffusion dynamics and gene expressions. Scaffold based or scaffoldless techniques for 3D cell cultures are rarely being compared in terms of the physical features of the microtissues produced. The biophysical properties of the microtissues cultured using scaffold based microencapsulation by flicking and scaffoldless liquid crystal (LC) based techniques were characterized. Flicking technique produced high yield and highly reproducible microtissues of keratinocyte cell lines in alginate microcapsules at approximately 350 ± 12 pieces per culture. However, microtissues grown on the LC substrates yielded at lower quantity of 58 ± 21 pieces per culture. The sizes of the microtissues produced using alginate microcapsules and LC substrates were 250 ± 25 μm and 141 ± 70 μm, respectively. In both techniques, cells remodeled into microtissues via different growth phases and showed good integrity of cells in field-emission scanning microscopy (FE-SEM). Microencapsulation packed the cells in alginate scaffolds of polysaccharides with limited spaces for motility. Whereas, LC substrates allowed the cells to migrate and self-stacking into multilayered structures as revealed by the nuclei stainings. The cells cultured using both techniques were found viable based on the live and dead cell stainings. Stained histological sections showed that both techniques produced cell models that closely replicate the intrinsic physiological conditions. Alginate microcapsulation and LC based techniques produced microtissues containing similar bio-macromolecules but they did not alter the main absorption bands of microtissues as revealed by the Fourier transform infrared spectroscopy. Cell growth, structural organization, morphology and surface structures for 3D microtissues cultured using both techniques appeared to be different and might be suitable for different applications.     

Generating human intestinal tissue from pluripotent stem cells in vitro

Here we describe a protocol for generating 3D human intestinal tissues (called organoids) in vitro from human pluripotent stem cells (hPSCs). To generate intestinal organoids, pluripotent stem cells are first differentiated into FOXA2(+)SOX17(+) endoderm by treating the cells with activin A for 3 d. After endoderm induction, the pluripotent stem cells are patterned into CDX2(+) mid- and hindgut tissue using FGF4 and WNT3a. During this patterning step, 3D mid- or hindgut spheroids bud from the monolayer epithelium attached to the tissue culture dish. The 3D spheroids are further cultured in Matrigel along with prointestinal growth factors, and they proliferate and expand over 1-3 months to give rise to intestinal tissue, complete with intestinal mesenchyme and epithelium comprising all of the major intestinal cell types. To date, this is the only method for efficiently directing the differentiation of hPSCs into 3D human intestinal tissue in vitro.     

Delivery of Human Adipose Stem Cells Spheroids into Lockyballs

Adipose stem cells (ASCs) spheroids show enhanced regenerative effects compared to single cells. Also, spheroids have been recently introduced as building blocks in directed self-assembly strategy. Recent efforts aim to improve long-term cell retention and integration by the use of microencapsulation delivery systems that can rapidly integrate in the implantation site. Interlockable solid synthetic microscaffolds, so called lockyballs, were recently designed with hooks and loops to enhance cell retention and integration at the implantation site as well as to support spheroids aggregation after transplantation. Here we present an efficient methodology for human ASCs spheroids biofabrication and lockyballs cellularization using micro-molded non-adhesive agarose hydrogel. Lockyballs were produced using two-photon polymerization with an estimated mechanical strength. The Young’s modulus was calculated at level 0.1362 +/-0.009 MPa. Interlocking in vitro test demonstrates high level of loading induced interlockability of fabricated lockyballs. Diameter measurements and elongation coefficient calculation revealed that human ASCs spheroids biofabricated in resections of micro-molded non-adhesive hydrogel had a more regular size distribution and shape than spheroids biofabricated in hanging drops. Cellularization of lockyballs using human ASCs spheroids did not alter the level of cells viability (p › 0,999) and gene fold expression for SOX-9 and RUNX2 (p › 0,195). The biofabrication of ASCs spheroids into lockyballs represents an innovative strategy in regenerative medicine, which combines solid scaffold-based and directed self-assembly approaches, fostering opportunities for rapid in situ biofabrication of 3D building-blocks.     

The role of actin filaments and microtubules in hepatocyte spheroid self-assembly

Cultured rat hepatocytes self-assemble into three-dimensional structures or spheroids that exhibit ultrastructural characteristics of native hepatic tissue and enhanced liver-specific functions. The spheroid formation process involves cell translocation and changes in cell shape, indicative of the reorganization of the cytoskeletal elements. To elucidate the function of the cytoskeleton, hepatocytes undergoing spheroid formation were treated with drugs that disrupt the different cytoskeletal components. Cytochalasin D, which targets the actin filaments, caused inhibition of spheroid formation. The role of microtubules in this process was assessed by incubating the cells with taxol or nocodazole. Perturbation of microtubules had minimal effects on spheroid assembly. Scanning electron micrographs showed no morphological differences between spheroids formed in control cultures and those formed in the presence of taxol or nocodazole. In addition, the effects of those agents on hepatocyte functions were investigated. Albumin secretion and cytochrome P450 2B1/2 activities of hepatocytes were comparable in spheroids formed in the presence of taxol or nocodazole to those formed in control cultures. The levels of these liver-specific activities were lower in cytochalasin D--treated cultures where only dispersed cells or cell clumps were found but spheroids had not found. Thus, hepatocytes require an intact actin network to self-assemble efficiently into functional tissue-like structures. Perturbation of the microtubule lattice does not impair the formation process. Events that transpire during hepatocyte spheroid self-assembly exhibit striking similarities to processes commonly observed in tissue morphogenesis. The results provide insight into the mechanisms that cells employ to organize into tissues and can contribute to our understanding of how to control the cellular assembly in tissue engineering and clinical applications.     

Recent advances in three-dimensional multicellular spheroid culture for biomedical research

Many types of mammalian cells can aggregate and differentiate into 3-D multicellular spheroids when cultured in suspension or a nonadhesive environment. Compared to conventional monolayer cultures, multicellular spheroids resemble real tissues better in terms of structural and functional properties. Multicellular spheroids formed by transformed cells are widely used as avascular tumor models for metastasis and invasion research and for therapeutic screening. Many primary or progenitor cells on the other hand, show significantly enhanced viability and functional performance when grown as spheroids. Multicellular spheroids in this aspect are ideal building units for tissue reconstruction. Here we review the current understanding of multicellular spheroid formation mechanisms, their biomedical applications, and recent advances in spheroid culture, manipulation, and analysis techniques.     

Three-dimensional co-culture of hepatocytes and stellate cells

Hepatocytes self-assemble in culture to form compacted spherical aggregates, or spheroids, that mimic the structure of the liver by forming tight junctions and bile canalicular channels. Hepatocyte spheroids thus resemble the liver to a great extent. However, liver tissue contains other cell types and has bile ducts and sinusoids formed by endothelial cells. Reproducing 3-D co-culture in vitro could provide a means to develop a more complex tissue-like structure. Stellate cells participate in revascularization after liver injury by excreting between hepatocytes a laminin trail that endothelial cells follow to form sinusoids. In this study we investigated co-culture of rat hepatocytes and a rat hepatic stellate cell line, HSC-T6. HSC-T6, which does not grow in serum-free spheroid medium, was able to grow under co-culture conditions. Using a three-dimensional cell tracking technique, the interactions of HSC-T6 and hepatocyte spheroids were visualized. The two cell types formed heterospheroids in culture, and HSC-T6 cell invasion into hepatocyte spheroids and subsequent retraction was observed. RT-PCR revealed that albumin and cytochrome P450 2B1/2 expression were better maintained in co-culture conditions. These three-dimensional heterospheroids provide an attractive system for in vitro studies of hepatocyte-stellate cell interactions.     

The liquid overlay technique is the key to formation of co-culture spheroids consisting of primary osteoblasts, fibroblasts and endothelial cells

Background aimsThe 3-dimensional (3-D) culture of various cell types reflects the in vivo situation more precisely than 2-dimensional (2-D) cell culture techniques. Spheroids as 3-D cell constructs have been used in tumor research for a long time. They have also been used to study angiogenic mechanisms, which are essential for the success of many tissue-engineering approaches. Several methods of forming spheroids are known, but there is a lack of systematic studies evaluating the performance of these techniques.MethodsWe evaluated the performance of the hanging drop technique, carboxymethyl cellulose technique and liquid overlay technique to form both mono- and co-culture spheroids consisting of primary osteoblasts, fibroblasts and endothelial cells. The performance of the three techniques was evaluated in terms of rate of yield and reproducibility. The size of the generated spheroids was determined systematically.ResultsThe liquid overlay technique was the most suitable for generating spheroids reproducibly. The rate of yield for this technique was between 60% and 100% for monoculture spheroids and 100% for co-culture spheroids. The size of the spheroids could be adjusted easily and precisely by varying the number of seeded cells organized in one spheroid. The formation of co-culture spheroids consisting of three different cell types was possible.ConclusionsOur results show that the most suitable technique for forming spheroids can vary from the chosen cell type, especially if primary cells are used. Co-culture spheroids consisting of three different cell types will be used to study angiogenic phenomena in further studies.     

Hepatocyte behavior within three-dimensional porous alginate scaffolds

A potential approach to facilitate the performance of implanted hepatocytes is to enable their aggregation and re-expression of their differentiated function prior to implantation. Here we examined the behavior of freshly isolated rat adult hepatocytes seeded within a novel three-dimensional (3-D) scaffold based on alginate. The attractive features of this scaffold include a highly porous structure (sponge-like) with interconnecting pores, and pore sizes with diameters of 100-150 microm. Due to their hydrophilic nature, seeding hepatocytes onto the alginate sponges was efficient. DNA measurements showed that the total cell number within the sponges did not change over 2 weeks, indicating that hepatocytes do not proliferate under these culture conditions. Nearly all seeded cells maintained viability, according to the MTT assay. Within 24 h post-seeding, small clusters of viable cells, were seen scattered within the sponge. More than 90% of the seeded cells participated in the aggregation; the high efficiency is attributed to the non-adherent nature of alginate. The spheroids had smooth boundaries and by day 4 in culture reached an average diameter of 100 microm, which is at the same magnitude of the sponge pore size. The cells appeared to synthesize fibronectin which was deposited on the spheroids. No laminin or collagen type IV were detected in the deposit. The 3-D arrangement of hepatocytes within the alginate sponges promoted their functional expression; within a week the cells secreted the maximal albumin secretion rate of 60 microg albumin/10(6) cells/day. Urea secretion rate did not depend on cell aggregation and was similar to that obtained when hepatocytes were cultured on collagen type I coated dishes (100 microg/10(6) cells/day). Our studies show that alginate sponges can provide a conducive environment to facilitate the performance of cultured hepatocytes by enhancing their aggregation.     

Long-term cryopreservation of reaggregated pancreatic islets resulting in successful transplantation in rats

Preservation of pancreatic islets for long-term storage of islets used for transplantation or research has long been a goal. Unfortunately, few studies on long-term islet cryopreservation (1 month and longer) have reported positive outcomes in terms of islet yield, survival and function. In general, single cells have been shown to tolerate the cryopreservation procedure better than tissues/multicellular structures like islets. Thus, we optimized a method to cryopreserve single islet cells and, after thawing, reaggregated them into islet spheroids. Cryopreserved (CP) single human islet cells formed spheroids efficiently within 3–5 days after thawing. Approximately 79% of islet cells were recovered following the single-cell cryopreservation protocol. Viability after long-term cryopreservation (4 weeks or more) was significantly higher in the CP islet cell spheroids (97.4 ± 0.4%) compared to CP native islets (14.6 ± 0.4%). Moreover, CP islet cell spheroids had excellent viability even after weeks in culture (88.5 ± 1.6%). Metabolic activity was 4–5 times higher in CP islet cell spheroids than CP native islets at 24 and 48 h after thawing. Diabetic rats transplanted with CP islet cell spheroids were normoglycemic for 10 months, identical to diabetic rats transplanted with fresh islets. However, the animals receiving fresh islets required a higher volume of transplanted tissue to achieve normoglycemia compared to those transplanted with CP islet cell spheroids. By cryopreserving single cells instead of intact islets, we achieved highly viable and functional islets after thawing that required lower tissue volumes to reverse diabetes in rats.     

A reliable tool to determine cell viability in complex 3-d culture: the acid phosphatase assay

Cell-based assays are more complex than cell-free test systems but still reflect a highly artificial cellular environment. Incorporation of organotypic 3-dimensional (3-D) culture systems into mainstream drug development processes is increasingly discussed but severely limited by complex methodological requirements. The objective of this study was to explore a panel of standard assays to provide an easy-handling, standardized protocol for rapid routine analysis of cell survival in multicellular tumor spheroid-based antitumor drug testing. Spheroids of 2 colon carcinoma cell lines were characterized for evaluation. One of the assay systems tested could reliably be used to determine cell viability in spheroids. The authors verified that the acid phosphatase assay (APH) is applicable for single spheroids in 96-well plates, does not require spheroid dissociation, and is linear and highly sensitive for HT29 and HCT-116 spheroids up to diameters of 650 microm and 900 microm, consisting of 40,000 and 80,000 cells, respectively. Treatment of HT29 and HCT-116 cells with 5-fluorouracil, Irinotecan, and C-1311 revealed critically reduced drug efficacies in 3-D versus monolayer culture, which is discussed in light of literature data. The experimental protocol presented herein is a small but substantial contribution to the establishment of sophisticated 3-D in vitro systems in the antitumor drug screening scenario.     

A quantitative analysis of the interplay of environment,neighborhood, and cell state in 3D spheroids

Cells react to their microenvironment by integrating external stimuli into phenotypic decisions via an intracellular signaling network. To analyze the interplay of environment, local neighborhood, and internal cell state effects on phenotypic variability, we developed an experimental approach that enables multiplexed mass cytometric imaging analysis of up to 240 pooled spheroid microtissues. We quantified the contributions of environment, neighborhood, and intracellular state to marker variability in single cells of the spheroids. A linear model explained on average more than half of the variability of 34 markers across four cell lines and six growth conditions. The contributions of cell‐intrinsic and environmental factors to marker variability are hierarchically interdependent, a finding that we propose has general implications for systems‐level studies of single‐cell phenotypic variability. By the overexpression of 51 signaling protein constructs in subsets of cells, we also identified proteins that have cell‐intrinsic and cell‐extrinsic effects. Our study deconvolves factors influencing cellular phenotype in a 3D tissue and provides a scalable experimental system, analytical principles, and rich multiplexed imaging datasets for future studies.