Three-dimensional Cell Based Bioreactors And Their Application In Screening And Analysis Of Bioactive Compounds From Chinese Medicinces

The drug discovery pipeline has produced many successful drug treatments and therapies. However, these successes have encountered some problems, such as high failure rates and costs. Moreover, they are also limited by a number of scientific and technical challenges to analyze drug candidates in a more rapid and accurate manner. One effective approach to solve these problems is to employ cell-based assays. The interaction of fluorescent proteins, microtubules, G-protein-coupled receptors, cell membranes and live cells with bioactive components has been reported. Live cell-based assays, using live cells as the bio-recognition elements, for drug screening are purposely considered to yield results that are more physiologically relevant when compared to biochemical assays and to provide meaningful information on drug compounds before costly animal tests. To provide efficient and economical methods for drug screening, micro-and nano-scale cell-based assay technologies have been studied in various stages of the drug discovery process. However, intrinsic drawbacks associated with conventional in vitro cellular tests using two-dimensional cultures, which lack three-dimensional (3D) scaffolds to support cell growth and proper tissue functions, cannot mimic in vivo microenvironments, and result in less accurate drugs'effects predictions. A more physiological in vitro model can provide more reliable data.3D cultures can mimic in vivo3D microenvironment of cells more actually. Thus,3D cell-based screening method would be more effective due to cellular responses would be more representative of those under in vivo conditions.Scaffolds offer several comparative advantages such as easier and faster to prepare, less resistance for diffusion of nutrients and wastes to and from cells respectively, and larger micro-scale pores enough to host multi-cellular aggregates. Various scaffolds have been prepared as templates for3D cell cultures among which composite scaffolds have attracted much attention as they exhibit superiorities of different materials and overcome drawbacks of single material.The ways of seeding cells onto scaffold influence cell spreading, proliferation and differentiation etc. Various static and dynamic seeding methods have been used for seeding cells onto scaffold. Compared with conventional static seeding and culture methods, recent studies have evidenced the positive impact of dynamic seeding and perfusion culture methods on the growth and survival of cells due to superior cell distribution and transfer of nutrients and gases which may affect cell proliferation in turn.It is of paramount importance to design bioreactors that ensure proper transport properties within a porous scaffold and to control the microenvironment surrounding cells. A vary type of packed-bed bioreactors have been used in dynamic seeding and perfusion culture of cells especially in hepatocyte high-density cultures. They have exhibited incomparable superiorities compared with static seeding and culture methods with regard to cell attachment, distribution, long-term survival and functions. However, packed-bed bioreactors, now available, cannot provide the essential environment for cells and are dependent on a CO2incubator which limits their applications. Moreover, there is no report on a pack-bed bioreactor for drug screening.In the light of these concerns, a new composite scaffold was fabricated and a packed-bed bioreactor undependable of CO2incubator was established for the screening and analysis of bioactive components interacted with3D cells from herbal medicines in this work.The thesis includes two parts. First part, chapter1, is general introduction while second part consisting of five chapters, is a research report.In chapter1, general introduction to drug screening models, high throughput drug screening and cell-based drug screening, and the superiority of3D cell cultures and commonly used methods for3D cell cultures, and design principles and category of3D cell bioreactors, and porous scaffold based3D cell bioreactors, commonly used biomaterials and methods for porous scaffold preparation, and the purpose, research content and key technology of this work were presented.In chapter2, a PLGA/hydroxyapatite/collagen porous scaffold was prepared and characterized, and the in vitro and in vivo biocompatibilities of the scaffold were evaluated. The influences of supercritical CO2saturation pressure, temperature and time on the pore structures of the PLGA/hydroxyapatite/collagen porous scaffold were investigated. Results showed that scaffolds with different pore size and porosity can be prepared by adjusting supercritical CO2saturation pressure, temperature and time. The chemical characterization showed that hydroxyapatite and collagen were homogeneously distributed in the scaffold. The in vitro biocompatibility assay showed that the PLGA/hydroxyapatite/collagen scaffold was suitable for MG-63cell growth and could promote MG-63cell proliferation. By implanting the PLGA/hydroxyapatite/collagen porous scaffold into the skin defects in symmetric sides of the rat back, the in vivo biocompatibility of the scaffold was investigated. HE staining showed that the inflammation caused by implanting of the scaffold could be eliminated after28days of surgery. ELS A results suggested that the contents of TNF-a, IL-1β and IL-6decreased gradually while IL-10increased gradually after surgery for both groups and the contents of TNF-a, IL-1β, IL-6and IL-10were similar to that in blank groups, suggesting that the inflammation elicited by the implantation of the scaffold was completely eliminated. Thus, it could be concluded that the scaffold had good in vitro and in vivo biocompatibilities and the scaffold would be widely used in tissue engineering.In chapter3, a PLGA-PEG-PLGA/hydroxyapatite/collagen porous scaffold was prepared and characterized, and the in vitro and in vivo biocompatibilities of this scaffold were evaluated. The influences of supercritical CO2saturation pressure, temperature and time on the pore structures of the PLGA-PEG-PLGA/hydroxyapatite/collagen porous scaffold were investigated. Results showed that scaffolds with different pore size and porosity can be prepared through adjusting supercritical CO2saturation pressure, temperature and time. The biocompatibility assay showed that the PLGA-PEG-PLGA/hydroxyapatite/collagen porous scaffold has good in vitro and in vivo biocompatibilities.In chapter4, a silk fibroin/hydroxyapatite/collagen porous scaffold was prepared and characterized, and the in vitro and in vivo biocompatibilities of this scaffold were evaluated. The influence of volume proportion of SF solution and collagen solution, the weight proportion of hydroxyapatite and NaCl, and the diameter of NaCl on the morphology the silk fibroin/hydroxyapatite/collagen porous scaffold were investigated. Results showed that scaffolds with different pore size and porosity can be prepared. The in vitro and in vivo biocompatibilities of the scaffold were evaluated. The biocompatibility assay showed that the silk fibroin/hydroxyapatite/collagen porous scaffold has good in vitro and in vivo biocompatibilities. In chapter5, a packed bed cell bioreactor coupled with HPLC/MS system for affinity screening of anticancer drugs was set up and used in bioactive components screening from Polygonum Cillinerve (Nakai) Ohwi (PCO). After interacting with live and fixed cells, the HPLC fingerprinting chromatograms of herbal medicine extract were compared to evaluate the binding properties of herbal components on cells. The interaction of model anticancer drugs (paclitaxel and resveratrol) and model non-anticancer drugs (ketoprofen and penicillin G) with model cancer cells (Lovo cells) demonstrated that this affinity screening system was feasible for drug screening. This affinity screening system was used to screen bioactive components from PCO extract, and two bioactive components, aristolochic acid A and aristolochic acid B, were screened out.In chapter6, a3D cell bioreactor for affinity screening of anticancer drugs was established and used in bioactive components screening from Sinopodophyllum hexandrum (Royle) Ying (SHY). Having significant difference (P<0.05) or not for model anticancer drugs and model non-anticancer drugs with model cancer cells demonstrated the feasibility of this method. Three compounds (quercetin-3-O-β-D-glucoside, kaempferol-3-O-β-D-glucoside and diphyllin) were considered non-specific binding with Lovo cells while other seven compounds, L-podophillotoxin-4-O-β-D-glucoside, quercetin, hexacosanoic acid, keaempferol, podophyllotoxin, podophyllone or isopicropodophyllone, and deoxypodophillotoxin or4'-demethylpodophyllotoxone were considered to be the bioactive components interacted with Lovo cells specifically, and their degrees of binding were32.6%±3.1%,29.3%±3.0%,15.2%±1.7%,57.1%±4.8%,68.9%±9.2%,63.6%±8.7%and46.3%±4.8%, respectively.In conclusion, three different porous scaffolds which lay the foundation for the estabilish of3D cell bioreactors were prepared, characterized and evaluated in this work. Two different3D bioreactors, undependable of CO2incubator, were established. The3D bioreactors developed in this work could mimic the in vivo microenvironment of drug-cells interaction. The3D bioreactors were respectively used for screening and analysis of bioactive components interacted with cancer cells specifically from Chinese medicines and some bioactive compounds were screened out. This affinity method could provide a potential tool for screening and analysis of bioactive components interacted specifically with cancer cells from the multi-component complex, as well as developing anticancer drug candidates from natural product libraries and herbal medicines.