Bioinspired 3D microprinted cell scaffolds: Integration of graph theory to recapitulate complex network wiring in lymph nodes.

Physical networks are ubiquitous in nature, but many of them possess a complex organizational structure that is difficult to recapitulate in artificial systems. This is especially the case in biomedical and tissue engineering, where the microstructural details of 3D cell scaffolds are important. Studies of biological networks-such as fibroblastic reticular cell (FRC) networks-have revealed the crucial role of network topology in a range of biological functions. However, cell scaffolds are rarely analyzed, or designed, using graph theory. To understand how networks affect adhered cells, 3D culture platforms capturing the complex topological properties of biologically relevant networks would be needed. In this work, we took inspiration from the small-world organization (high clustering and low path length) of FRC networks to design cell scaffolds. An algorithmic toolset was created to generate the networks and process them to improve their 3D printability. We employed tools from graph theory to show that the networks were small-world (omega factor, ω = -0.10 ± 0.02; small-world propensity, SWP = 0.74 ± 0.01). 3D microprinting was employed to physicalize networks as scaffolds, which supported the survival of FRCs. This work, therefore, represents a bioinspired, graph theory-driven approach to control the networks of microscale cell niches.

A Hydrogel-Integrated Culture Device to Interrogate T Cell Activation with Physicochemical Cues.

The recent rise of adoptive T cell therapy (ATCT) as a promising cancer immunotherapy has triggered increased interest in therapeutic T cell bioprocessing. T cell activation is a critical processing step and is known to be modulated by physical parameters, such as substrate stiffness. Nevertheless, relatively little is known about how biophysical factors regulate immune cells, such as T cells. Understanding how T cell activation is modulated by physical and biochemical cues may offer novel methods to control cell behavior for therapeutic cell processing. Inspired by T cell mechanosensitivity, we developed a multiwell, reusable, customizable, two-dimensional (2D) polyacrylamide (PA) hydrogel-integrated culture device to study the physicochemical stimulation of Jurkat T cells. Substrate stiffness and ligand density were tuned by concentrations of the hydrogel cross-linker and antibody in the coating solution, respectively. We cultured Jurkat T cells on 2D hydrogels of different stiffnesses that presented surface-immobilized stimulatory antibodies against CD3 and CD28 and demonstrated that Jurkat T cells stimulated by stiff hydrogels (50.6 ± 15.1 kPa) exhibited significantly higher interleukin-2 (IL-2) secretion, but lower proliferation, than those stimulated by softer hydrogels (7.1 ± 0.4 kPa). In addition, we found that increasing anti-CD3 concentration from 10 to 30 µg/mL led to a significant increase in IL-2 secretion from cells stimulated on 7.1 ± 0.4 and 9.3 ± 2.4 kPa gels. Simultaneous tuning of substrate stiffness and stimulatory ligand density showed that the two parameters synergize (two-way ANOVA interaction effect: p < 0.001) to enhance IL-2 secretion. Our results demonstrate the importance of physical parameters in immune cell stimulation and highlight the potential of designing future immunostimulatory biomaterials that are mechanically tailored to balance stimulatory strength and downstream proliferative capacity of therapeutic T cells.

Rethinking Cancer Immunotherapy by Embracing and Engineering Complexity.

The meteoric rise of cancer immunotherapy in the past decade has led to promising treatments for a number of hard-to-treat malignancies. In particular, adoptive T cell therapy has recently reached a major milestone with two products approved by the US FDA. However, the inherent complexity of cell-based immunotherapies means that their manufacturing time, cost, and controllability limit their effectiveness and geographic reach. One way to address these issues may lie in complementing the dominant, reductionistic mentality in modern medicine with complex systems thinking. In this opinion article, we identify key concepts from complexity theory to address manufacturing challenges in cell-based immunotherapies and raise the possibility of a unifying framework upon which future bioprocessing strategies may be designed.

Articular cartilage degeneration following anterior cruciate ligament injury: a comparison of surgical transection and noninvasive rupture as preclinical models of post-traumatic osteoarthritis.

OBJECTIVE: Post-traumatic osteoarthritis (PTOA) is commonly studied using animal models. Surgical ACL transection is an established model, but noninvasive models may mimic human injury more closely. The purpose of this study was to quantify and compare changes in 3D articular cartilage (AC) morphology following noninvasive ACL rupture and surgical ACL transection. METHODS: Thirty-six rats were randomized to uninjured control, noninvasive ACL rupture (Rupture), and surgical ACL transection (Transection), and 4 and 10 week time points (n = 6 per group). Contrast-enhanced micro-computed tomography (CE-µCT) was employed for AC imaging. Femoral and tibial AC were segmented and converted into thickness maps. Compartmental and sub-compartmental AC thickness and surface roughness (Sa) were computed. OARSI histologic scoring was performed. RESULTS: In both injury groups, zones of adjacent thickening and thinning were evident on the medial femoral condyle, along with general thickening and roughening of femoral and tibial AC. The posterior tibia exhibited drastic thickening and surface degeneration, and this was worse in Transection. Both injury groups had increased AC thickness and Sa compared to Control at both time points, and Transection exhibited significantly higher Sa in every tibial compartment compared to Rupture. Histologic score was elevated in both groups, and the medial femur exhibited the most severe histologic degeneration. CONCLUSIONS: This is the first 3D quantification of preclinical AC remodeling after ACL injury. Both injury models induced similar changes in AC morphology, but Transection exhibited higher tibial Sa and a greater degree of posterior tibial degeneration. We conclude that AC degeneration is a time-, compartment-, and injury-dependent cascade.

Surface roughness and thickness analysis of contrast-enhanced articular cartilage using mesh parameterization.

OBJECTIVE: Articular cartilage (AC) morphology is an important metric for characterizing degeneration. We propose a novel morphologic analysis using mesh parameterization, enabling the use of surface roughness and thickness metrics to characterize degeneration in a rodent model of post-traumatic osteoarthritis. METHODS: Six rats underwent anterior cruciate ligament transection (ACL-T) and six were controls (Control). At 4-weeks, femora and tibiae were harvested and imaged using contrast-enhanced micro-computed tomography (µCT). Cartilage surfaces were manually outlined, and 2-dimensional thickness maps were generated using mesh parameterization and analyzed by thickness and surface roughness (Sa). The parameterization technique was validated against the direct distance transform (DDT) and histologic AC thickness from sagittal Safranin-O/Fast-Green sections. Parameterization and DDT measurements were also validated using known, virtual shapes with zero, one, and two planes of curvature. RESULTS: Parameterization had 0.00-6.26% error and DDT had 5.06-12.02% error in determining thicknesses of known shapes. Parameterization thickness correlated highly to DDT thickness (femur: r = 0.978, P < 0.001; tibia: r = 0.992, P < 0.001) and histologic thickness (femur: r = 0.952, P < 0.001; tibia: r = 0.959, P < 0.001). Thickness maps enabled visualization and quantification of AC degeneration. ACL-T samples displayed general thickening of cartilage, with adjacent regions of thickening and thinning on the medial femoral condyle. Compared to Control, ACL-T thickness was higher in the whole femur, whole tibia, and all compartments and sub-compartments. Sa was higher in the whole femur and medial and lateral condyle, and the whole tibia and medial and lateral plateau. The largest increases in Sa were observed on the medial femoral condyle. CONCLUSIONS: Cartilage analysis using parameterization effectively characterized early degeneration in AC, including sub-compartmental thickening/thinning, and is a powerful tool for assessing degeneration in preclinical osteoarthritis.

Subchondral and epiphyseal bone remodeling following surgical transection and noninvasive rupture of the anterior cruciate ligament as models of post-traumatic osteoarthritis.

OBJECTIVE: Animal models are frequently used to study post-traumatic osteoarthritis (PTOA). A common anterior cruciate ligament (ACL) injury model is surgical transection, which may introduce confounding factors from surgery. Noninvasive models could model human injury more closely. The purpose of this study was to compare subchondral and epiphyseal trabecular bone remodeling after surgical transection and noninvasive rupture of the ACL. METHODS: Thirty-six rats were randomized to an uninjured control, surgical transection (Transection), or noninvasive rupture (Rupture). Animals were randomized to 4 or 10 week time points (n = 6 per group). Micro computed tomography (µCT) imaging was performed with an isotropic voxel size of 12 µm. Subchondral and epiphyseal bone was segmented semi-automatically, and morphometric analysis was performed. RESULTS: Transection caused a greater decrease in subchondral bone volume fraction (BV/TV) than Rupture in the femur and tibia. Rupture had greater subchondral bone tissue mineral density (TMD) at 4 and 10 weeks in the femur and tibia. Subchondral bone thickness (SCB.Th) was decreased in the femur in Transection only. Epiphyseal BV/TV was decreased in Transection only, and Rupture exhibited increased femoral epiphyseal TMD compared to both Control and Transection. Rupture exhibited greater femoral epiphyseal trabecular thickness (Tb.Th.) compared to Control and Transection at 4 weeks, and both Rupture and Transection had increased femoral epiphyseal Tb.Th. at 10 weeks. Epiphyseal trabecular number (Tb.N) was decreased in both injury groups at both time points. Femoral and tibial epiphyseal structure model index (SMI) increased in both groups. CONCLUSIONS: The two injury models cause differences in post-injury bone morphometry, and surgical transection may be introducing confounding factors that affect downstream bony remodeling.

Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review.

Once damaged, articular cartilage has very little capacity for spontaneous healing because of the avascular nature of the tissue. Although many repair techniques have been proposed over the past four decades, none has sucessfully regenerated long-lasting hyaline cartilage tissue to replace damaged cartilage. Tissue engineering approaches, such as transplantation of isolated chondrocytes, have recently demonstrated tremendous clinical potential for regeneration of hyaline-like cartilage tissue and treatment of chondral lesions. As such a new approach emerges, new important questions arise. One of such questions is: what kinds of biomaterials can be used with chondrocytes to tissue-engineer articular cartilage? The success of chondrocyte transplantation and/or the quality of neocartilage formation strongly depend on the specific cell-carrier material. The present article reviews some of those biomaterials, which have been suggested to promote chondrogenesis and to have potentials for tissue engineering of articular cartilage. A new biomaterial, a chitosan-based polysaccharide hydrogel, is also introduced and discussed in terms of the biocompatibility with chondrocytes.

Vascular cell responses to polysaccharide materials: in vitro and in vivo evaluations.

Chitosan has shown promise as a structural material for a number of tissue engineering applications. Similarly the glycosaminoglycans (GAGs) and their analogs have been known to exert a variety of biological activities. In this study we evaluated the potential of GAG-chitosan and dextran sulfate (DS)-chitosan complex materials for controlling the proliferation of vascular endothelial (EC) and smooth muscle cells (SMC). GAG-chitosan complex membranes were generated in vitro and seeded with human ECs or SMCs for culture up to 9d. In addition, porous chitosan and GAG-chitosan complex scaffolds were implanted subcutaneously in rats to evaluate the in vivo response to these materials. The results indicated that while chitosan alone supported cell attachment and growth, GAG-chitosan materials inhibited spreading and proliferation of ECs and SMCs in vitro. In contrast, DS-chitosan surfaces supported proliferation of both cell types. In vivo, heparin-chitosan and DS-chitosan scaffolds stimulated cell proliferation and the formation of a thick layer of dense granulation tissue. In the case of heparin scaffolds the granulation layer was highly vascularized. These results indicate that the GAG-chitosan materials can be used to modulate the proliferation of vascular cells both in vitro and in vivo.

GAG-augmented polysaccharide hydrogel: a novel biocompatible and biodegradable material to support chondrogenesis.

The quality of articular cartilage engineered using a cell-polymer construct depends, in part, on the chemical composition of the biomaterial and whether that biomaterial can support the chondrocytic phenotype. Acknowledging the supportive influence of tissue-specific matrix molecules on the chondrocytic phenotype, we have combined chondroitin sulfate-A (CSA) and chitosan, a glycosaminoglycan (GAG) analog, to develop a novel biomaterial to support chondrogenesis. Chitosan is a polycationic repeating monosaccharide of beta-1,4-linked glucosamine monomers with randomly located N-acetyl glucosamine units. Chitosan may be combined with the polyanionic CSA such that ionic crosslinking results in hydrogel formation. Bovine primary articular chondrocytes, when seeded onto a thin layer of CSA-chitosan, form discrete, focal adhesions to the material and maintain many characteristics of the differentiated chondrocytic phenotype, including round morphology, limited mitosis, collagen type II, and proteoglycan production. Our findings suggest CSA-chitosan may be well suited as a carrier material for the transplant of autologous chondrocytes or as a scaffold for the tissue engineering of cartilage-like tissue.

Maintenance of CD34 expression during proliferation of CD34+ cord blood cells on glycosaminoglycan surfaces.

Recent studies have indicated that glycosaminoglycan (GAG) interactions with hematopoietic progenitors play a significant role in the regulation of hematopoiesis. However, the details of these interactions are not clear. In this study, we examined the role of soluble and immobilized GAGs in the proliferation of CD34+ cells. Chitosan, a cationic polysaccharide, was used to immobilize GAGs in ionic complex membranes. The GAGs studied were heparin, hyaluronate, and chondroitin sulfates A, B, and C. CD34-enriched umbilical cord blood cells were seeded onto tissue culture plates coated with the GAG-chitosan complex membranes. Cultures were maintained in medium supplemented with stem cell factor and interleukin 3 for up to six weeks, during which total and CD34+ cell numbers were determined by flow cytometry. Total cell number expansion ranged from 25-fold to 40-fold after six weeks. However, only heparin and chondroitin sulfate B (CSB) surfaces retained a significant CD34+ fraction. All other surfaces exhibited declines in CD34 expression, with negligible CD34+ percentages remaining after four weeks. In contrast, heparin and CSB surfaces exhibited CD34+ fractions as high as 90% after four weeks. GAG desorption studies indicated that the observed effects were partly mediated by desorbed GAGs in a concentration dependent manner. Subsequent studies showed that sustained high (160 microg/ml) heparin levels had toxic effects, while the same concentration of CSB exhibited more rapid early proliferation of CD34+ cells. In conclusion, this culture system has demonstrated the ability to produce simultaneous proliferation and CD34+ cell enrichment of a partially purified cord blood population by controlling the nature and levels of GAG moieties to which the cells are exposed. The results indicate that specific GAGs can significantly influence the growth and differentiation characteristics of cultured CD34+ cells.

Porous chitosan scaffolds for tissue engineering.

The wide array of tissue engineering applications exacerbates the need for biodegradable materials with broad potential. Chitosan, the partially deacetylated derivative of chitin, may be one such material. In this study, we examined the use of chitosan for formation of porous scaffolds of controlled microstructure in several tissue-relevant geometries. Porous chitosan materials were prepared by controlled freezing and lyophilization of chitosan solutions and gels. The materials were characterized via light and scanning electron microscopy as well as tensile testing. The scaffolds formed included porous membranes, blocks, tubes and beads. Mean pore diameters could be controlled within the range 1-250 microm, by varying the freezing conditions. Freshly lyophilized chitosan scaffolds could be treated with glycosaminoglycans to form ionic complex materials which retained the original pore structure. Chitosan scaffolds could be rehydrated via an ethanol series to avoid the stiffening caused by rehydration in basic solutions. Hydrated porous chitosan membranes were at least twice as extensible as non-porous chitosan membranes, but their elastic moduli and tensile strengths were about tenfold lower than non-porous controls. The methods and structures described here provide a starting point for the design and fabrication of a family of polysaccharide based scaffold materials with potentially broad applicability.

Rapid hepatocyte spheroid formation: optimization and long-term function in perfused microcapsules.

Enhancement of cell-cell interactions and, hence, long-term function in liver support systems can be effected by controlling the diameters of hepatocyte aggregates or spheroids. In this study, primary rat hepatocytes were induced to rapidly form spheroids using an intermittent settling and agitation protocol. The cells were seeded into albumin coated flasks at densities ranging from 80,000 to 520,000 cells/cm2. Hepatocytes were resuspended for 15 sec at 20-min intervals by placing the flasks on a timer controlled linear shaker. At time points ranging from 8 to 24 hr, hepatocyte aggregates were imaged via light microscopy. Mean spheroid diameter and shape factor were determined using computer analysis of captured images. Spheroid diameter could be controlled within the range of 60 to 240 microns. For long-term evaluation, spheroids were microencapsulated and cultured for 21 days under perfusion conditions. Encapsulated spheroids secreted albumin at rates comparable to collagen sandwich control cultures for at least 14 days, with peak rates (approximately 80 microns/day/10(6) cells) exhibited after culture medium changes. The results show that controlled, high efficiency hepatocyte aggregation can be accomplished in as little as 8 hr, and that the encapsulated spheroids exhibit long-term in vitro function.

Extracorporeal plasma perfusion of cultured hepatocytes: effect of intermittent perfusion on hepatocyte function and morphology.

The most promising approaches to developing a temporary bioartificial liver support system involve incorporating cultured primary hepatocytes into an extracorporeal perfusion device. As a result, it is important to characterize both the phenotypic response of these cells during extracorporeal perfusion and the critical factors involved in maintaining differentiated cell function over extended periods of perfusion. In this study, hepatocytes cultured in a collagen sandwich configuration were connected to a rat via a hollow fiber plasma separator and perfused with plasma on line. Perfusions were either continuous for 48 hr or intermittent for up to 174 hr with 6 hr per day of extracorporeal plasma perfusion alternating with 18 hr of culture medium perfusion. During perfusion cell morphology was continuously monitored by time-lapse video microscopy. After the procedure, hepatocytes were returned to static culture and function was evaluated by measuring the rates of urea synthesis daily for 7 days. During plasma perfusion all hepatocytes accumulated cytoplasmic lipid droplets in a time dependent manner. Urea synthesis was maintained at initial levels for up to 20 hr of continuous plasma perfusion. However, urea synthesis rates were reduced by 31 and 52% after 30 and 48 hr of continuous plasma exposure, respectively. With intermittent perfusions, as well as with control cells perfused with culture medium only, urea synthesis rates did not decrease for at least 78 hr of total perfusion. There was no difference between the urea synthesis rates after 48 hr of cumulative plasma exposure time between cells subjected to continuous and intermittent plasma perfusion. These results suggest that cultured hepatocytes may be exposed to plasma for at least 20 hr with no significant reduction in liver-specific function. Furthermore, an intermittent plasma perfusion schedule can be used to divide the useful plasma perfusion time over several days with no adverse effects on cell function.

Effects of plasma exposure on cultured hepatocytes: Implications for bioartificial liver support.

In order to examine their potential for use in a bioartificial liver, hepatocytes maintained in a collagen sandwich configuration were cultured for 9 days in heparinized rat plasma. The cells exhibited a progressive accumulation of cytoplasmic lipid droplets which proved to be mainly triglyceride (TG). The rate of TG accumulation correlated with the free fatty acid (FFA) content of the plasma. Removal of FFA and TG from plasma by ether extraction significantly reduced the rate and extent of TG accumulation. A smaller reduction in the rate and extent of TG accumulation was observed when cells were maintained in an oxygen enriched environment. The lipid accumulation suppressed urea synthesis, but clearance of the drug diazepam, although constitutively depressed in plasma, appeared unaffected by the accumulation. The functional and morphological effects of plasma exposure could be fully reversed after at least 6 days of plasma exposure by returning the cells to culture medium.The results indicate that elevated FFA in plasma induces lipid accumulation, which inhibits urea synthesis in cultured hepatocytes. This suggests that estimates of the cell number needed for effective liver support should not be based upon function measurements conducted in culture media. Furthermore, optimization of bioartificial liver support device use may have to be governed by the need to limit the plasma exposure of cultured hepatocytes. However, the highly responsive nature of these cultures and the reversibility of the plasma effects suggest that the collagen sandwich culture system is a promising foundation for the development of an effective bioartificial liver support system. (c) 1996 John Wiley & Sons, Inc.

The activity of cytochrome P450IA1 in stable cultured rat hepatocytes.

In this study, a stable, differentiated culture format (primary hepatocytes cultured between two layers of collagen gel) was used to study the effect of the inducer 3,3',4,4'-tetrachlorobiphenyl (TCB) on the activity of cytochrome P50IA1. P450IA1 (ethoxyresorufin O-deethylase) enzymatic activity was measured using the rate of conversion of ethoxyresorufin (ER) to resorufin (R). After 14 days of induction with 10(-6), M TCB, hepatocytes in the double collagen gel configuration exhibited a maximum activity of 180.3 +/- 46.8 pmol R/mug, DNA/hr (3.6 +/- 0.9 nmol R/10(6), cells/hr) compared with 34.9 +/- 3.8 pmol R/mug DNA/hr for cells cultured on a single layer of gel. At a TCB level of 10(-5), m, the P450IA1 activity in the double-gel configuration peaked at 220.8 +/- 37.0 pmol R/mug DNA/hr. Cessation of 10(-6), M TCB induction produced a decrease in activity to 25.8 +/- 4.1 pmol R/mug DNA/hr within 4 hr. Subsequent re-application of the inducer caused an increase in activity to 76.5 +/- 11.1 pmolR/mug DNA/hr within 6 hr, reaching a maximal value of 131.0 +/- 38.6 pmol R/mug DNA/hr within 12 hr. Since TCB is rapidly metabolized by hepatocytes, a continuous perfusion culture system was developed to examine the effect of exposure to a constant level of TCB. Continuous perfusion of the cells with 10(-8) or 10(-7), M TCB, resulted in activities significantly higher than those of cultures induced by daily application of induction medium. A mechanistic model of TCB-dependent induction of P450IA1 was developed using kinetic parameters estimated from static culture data. The model accurately predicted cyclic variations in P450IA1 activity in static culture, and the steady-state activity level of perfusion cultures. This work describes procedures for exposing stable hepatocyte cultures to either continuous or declining levels of consumable inducers and for measuring the activity of cytochrome P50IA1 in cultured hepatocytes by a non-invasive method.

Performance of plasma-perfused, microencapsulated hepatocytes: prospects for extracorporeal liver support.

The growing success of liver transplantation and the shortage of donor livers has turned attention to the possibility of utilizing hepatocytes within artificial liver support systems to allow time for donor livers to become available and to improve the condition of patients with hepatic failure. This study evaluated encapsulated hepatocytes, a technology which might allow the possibility of using xenogenic or human hepatoma cells. Rabbit hepatocytes were encapsulated using the ionic polysaccharides carboxymethylcellulose, chondroitin sulfate A, chitosan, and polygalacturonic acid. Encapsulated cells were maintained in perfusion culture for at least 6 days in heparinized, normal human plasma or in a defined culture medium. Parallel cultures of plated hepatocytes were also conducted. The metabolic capability of the cells was evaluated by following the rates of urea, albumin, and transferrin synthesis and the transformation rate of the drug antipyrine. Protein synthesis and ureogenesis in plasma were depressed from the levels expressed in defined culture medium. Drug detoxification as measured by antipyrine metabolism appeared to be enhanced in plasma. We conclude that encapsulated rabbit hepatocytes retain significant levels of function for at least 6 days of perfusion with human plasma, suggesting the feasibility of this technology as a potential method of short-term liver support.

Complex coacervate microcapsules for mammalian cell culture and artificial organ development.

A number of combinations of anionic and cationic polymers, the majority being polysaccharides, were screened to determine their suitability for the development of alternative microcapsule formulations capable of supporting cells. The capsules were taken through a limited optimization and then evaluated on the bases of rupture strength, permeability to albumin, and ability of their components to promote the attachment, aggregation, and function of encapsulated rabbit hepatocytes. The widely used alginate-polylysine capsules were employed as a comparative standard in all tests. A number of the new formulations compared favorably with the standard, and some exhibited superior performance in specific areas. Hepatocyte function, as evaluated by the rate of urea synthesis, showed no significant differences between formulations over a 24-h test period. One formulation, composed of the polysaccharides (carboxymethyl)cellulose, chondroitin sulfate A, chitosan, and polygalacturonate, was found to be superior to alginate-polylysine capsules in the areas investigated and supported the long-term survival and growth of liver endothelial cells.

Microencapsulated hepatocytes. Prospects for extracorporeal liver support.

To assess the potential for encapsulated hepatocytes as a bioartificial liver support system, rabbit hepatocytes were encapsulated within multicomponent capsules using a complex coacervation technique, and cultured both on plates and in a perfusion reactor. The urea synthesis rate and antipyrine and diazepam degradation rates were evaluated in each system over a 10 day period, and compared with standard plate-cultured hepatocyte efficacy. Urea synthesis rates were significantly higher in the perfusion cultures than in either of the plate culture environments, whereas drug degradation rates were not significantly different in any of the systems.