3D in vitro bioengineered tumors based on collagen I hydrogels

Abstract

Cells cultured within a three-dimensional (3D) in vitro environment have the ability to acquire phenotypes and respond to stimuli analogous to in vivo biological systems. This approach has been utilized in tissue engineering and can also be applied to the development of a physiologically relevant in vitro tumor model. In this study, collagen I hydrogels cultured with MDA-MB-231 human breast cancer cells were bioengineered as a platform for in vitro solid tumor development. The cell–cell and cell-matrix interactions present during in vivo tissue progression were encouraged within the 3D hydrogel architecture, and the biocompatibility of collagen I supported unconfined cellular proliferation. The development of necrosis beyond a depth of ~150–200 μm and the expression of hypoxia-inducible factor (HIF)-1α were demonstrated in the in vitro bioengineered tumors. Oxygen and nutrient diffusion limitations through the collagen I matrix as well as competition for available nutrients resulted in growing levels of intra-cellular hypoxia, quantified by a statistically significant (p < 0.01) upregulation of HIF-1α gene expression. The bioengineered tumors also demonstrated promising angiogenic potential with a statistically significant (p < 0.001) upregulation of vascular endothelial growth factor (VEGF)-A gene expression. In addition, comparable gene expression analysis demonstrated a statistically significant increase of HIF-1α (p < 0.05) and VEGF-A (p < 0.001) by MDA-MB-231 cells cultured in the 3D collagen I hydrogels compared to cells cultured in a monolayer on two-dimensional tissue culture polystyrene. The results presented in this study demonstrate the capacity of collagen I hydrogels to facilitate the development of 3D in vitro bioengineered tumors that are representative of the pre-vascularized stages of in vivo solid tumor progression.

The Effect of Chick Embryo Cerebro-Spinal Fluid in Microwave Irradiated Collagen Guide Channel on Sciatic Nerve Regeneration in Rat

“…The supernatant, containing the collagen-I that was stored in sterile universal bottles in 15ml aliquots at 4°C until use. Collagen protein concentration was measured by dry weight and averaging the concentration values by evaporating out the solvent from 0.25, 0.5, and 1.0 ml samples in a 110 °C oven for 2 h. In order to sterilize the collagen I, chloroform (10% of the volume of collagen) was added to bottom of the bottle beneath the collagen-I solution and allowed to rest overnight at 4°C [13]. We then aseptically removed the collagen solution.…”

The effect of cerebro-spinal fluid in collagen guide channel on sciatic nerve regeneration in rat

“…The supernatant was removed, and averaging the 3% collagen solution was obtained. In order to sterilize the collagen I, chloroform (10% of the volume of collagen) was layered beneath the collagen solution and allowed to rest for 24 h at 4 °C (28). The solution was immersed in demonized water, pH to 7.4 with NaOH and PBS on ice and mixed thoroughly with a pipette (9).…”

“…The collagen nerve guide is flexible and strong enough to hold a suture and the dimensions of the guide are easily controlled (2), a secondary surgical site is not needed to obtain the collagen guide channel, and there is no donor site morbidity (1). In addition, the collagen can be obtained in large quantities, being inexpensive, sterilized and easily stored (1, 25,28). Collagen molecules can easily trap neurotrophic factors and collagen channels are able to adsorb cell adhesion molecules (17).…”

In VitroAngiogenesis Induced by Tumor-Endothelial Cell Co-Culture in Bilayered, Collagen I Hydrogel Bioengineered Tumors

“…29 MDA-MB-231 and MCF7 cells were cultured separately in an 8 mg/mL collagen solution at a seeding density of 5 · 10 6 cells/mL. The tumor cell-collagen suspension was pipetted into 12-well costar transwell inserts with 0.4 mm pore polyester membranes (Corning Incorporates, Corning, NY).…”

“…Furthermore, most co-culture angiogenesis models have been unsuccessful in achieving invasive sprouting without the contribution of exogenous angiogenic growth factors 26,27 or fibroblasts. [23][24][25]28 To address this deficiency, we introduce an in vitro angiogenesis model based on microvascular endothelial cells cultured as a confluent monolayer on the surface of previously characterized pro-angiogenic bioengineered tumors 29 in the absence of exogenous angiogenic growth factors. These in vitro tumors, composed of MDA-MB-231 breast cancer cells cultured within a bulk collagen I hydrogel, have been shown to exhibit phenotypic characteristics that are ideal for encouraging an in vivo-like angiogenic response, including the development of a necrotic core, intracellular levels of hypoxia, and subsequent up-regulation of VEGFA gene expression.…”