The purpose of this study was to rationally design animal component free, chemically defined serum free media (ACF-CD-SFM) for MRC-5 and Vero cells while adhering to the Quality by Design guidelines. This was achieved by using the Modified Vero Serum Free Medium (MVSFM) as the basal formulation and supplementing it with various combinations of growth factors (LONG® EGF, LONG® R3 IGF-I, rTransferrin, bFGF, TGF-3 and PDGF-AA), lipids (linoleic acid, cholesterol, and dexamethasone), lipid precursors (ethanolamine and phosphoethanolamine) and vitamins (all-trans retinoic acid, -tocopherol and ascorbic acid). Media development was achieved by conducting a series of steps using different experimental methodologies with the end goal of satisfying the requirements of each cell line. MRC-5 and Vero cells were each cultured in specific media containing unique concentrations of supplements that were prepared according to the different statistical design methodologies. The original objective was to create a SFM, however due to the stringent nutritious requirements of anchorage dependent cell lines, only a reduction to 0.5% FBS was achieved. For MRC-5 cells, the one-factor-at-a-time (OFAT) generated the Prototype + 0.5% FBS medium. The Definitive Screening Design (DSD) gave rise to the Delta 1 + 0.5% FBS, which was the optimum medium formulation for MRC-5 cells as it had comparable cell yields to DMEM + 10 % FBS. This result was confirmed by the Genetic Algorithms-Hill Climbing (GA-HC) method. In the case of Vero cells, the OFAT and the DSD confirmed that MVSFM + 0.5 % FBS was the most optimal formulation. The morphology in both media for both cell lines was comparable to that in DMEM-10% FBS. It was concluded that the DSD method successfully achieved a reduction of the serum concentration from 10% to 0.5% FBS. / October 2017

Mouse embryo cells cultured in vitro in serum-supplemented media undergo growth crisis, resulting in the loss of genomically normal cells prior to the appearance of established, aneuploid cell lines. I used the technique of serum-free cell culture to develop a serum-free mouse embryo (SFME) cell line in which serum was replaced by a set of defined supplements. SFME cells, cultured in a nutrient medium supplemented with insulin, transferrin, epidermal growth factor (EGF), high-density lipoprotein (HDL), and fibronectin, have maintained a diploid karyotype with no detectable chromosomal abnormalities for more than 200 generations. The cells did not undergo growth crisis and remain in culture today. SFME cells were dependent on EGF for survival and were reversibly growth inhibited by serum or platelet-free plasma. Treatment of SFME cells with serum or transforming growth factor beta led to the appearance of glial fibrillary acid protein (GFAP), a specific marker for astrocytes, identifying SFME cells as proastroblasts. Following the derivation of SFME cells my research focussed on (1) defining more precisely the growth response of SFME cells to medium supplements, (2) investigating the relationship between the nonsenescent nature of SFME cells and their responses to serum and EG1., and (3) applying the serum-free cell culture methods to the multipassage culture of human embryonic astrocytes. SFME cells in serum-containing medium arrested in the G1 phase of the cell cycle with greatly reduced DNA replication activity. A portion of the inhibitory activity of serum was extracted by charcoal, a procedure that removed steroid and thyroid hormones. However, the effect of serum on untransformed SFME cells could not be prevented by addition of antiglucocorticoid, and ras-transformed clones of SFME cells, which are not growth inhibited by serum, retained inhibitory responses to glucocorticoid and thyroid hormone T3. These results suggest that glucocorticoid or thyroid hormones may contribute to the inhibitory activity of serum on SFME cells, but additional factors are involved. SFME cell death resulting from EGF deprivation exhibited characteristics associated with apoptosis or programmed cell death. Ultrastructural analysis showed cells became small and vacuolated, with pyknotic nuclei. The cultures contained almost exclusively G1- phase cells. Chromatin exhibited a pattern of degradation into oligonucleosome-length fragments generating a regularly spaced ladder. I applied the serum-free approach used to derive SFME cells to the multipassage culture of human embryonic astrocytes. Cells were cultured in nutrient medium supplemented with insulin, transferrin, EGF, HDL, fibronectin, basic fibroblast growth factor and heparin. Cultures were maintained for a maximum of 70 population doublings before proliferation ceased. The cells synthesized GFAP. / Graduation date: 1992

The objective of this work was the investigation of nanofiltration as a potential avenue to fractionate protein hydrolysates and produce protein hydrolysate fractions with stimulating bioactivity for the development of a supplement for a serum-free media. Mammalian cell culture is widely used for the production of therapeutic proteins such as antibodies, interleukins, and vaccines because of the ability of mammalian cells to glycosylate proteins. A complex media with the addition of serum is often required to meet the requirements of the cells. Although serum is a supplement that provides different proteins such as growth factors and hormones, serum has several disadvantages such as high cost, difficulty of downstream processing due to its high protein content and the possibility of microbiological contamination. Protein hydrolysates from plant, animal, or yeast cells contain a complex mixture of peptides and amino acids and have been shown to enhance growth of certain mammalian cell lines cultured in serum-free media. To fractionate peptide mixtures, nanofiltration was investigated in this study. Nanofiltration is a pressure driven membrane separation process based on size and charge. The investigation of pH and NaCl on the filtration performance for two different nanofiltration membranes (HL membrane and G-10 membrane) was achieved using a 24 factorial design. The total peptide concentration, the antioxidant activity, and organic and inorganic content were analyzed in the permeate and retentate fraction. The fractions were also tested for their enhanced growth ability and the specific -interferon productivity with CHO cells. Furthermore the retentate and permeate fractions were analyzed by reversed phase-HPLC to characterize the peptide and free amino acid distribution profile. Through the factorial design, the membrane type was shown to have a significant effect on the filtration performance for both yeast extract and Primatone. A significant difference, but similar for both feed sources, was observed for the total peptide transmission with around 10% for the HL membrane and around 30% for the G-10 membrane. The average permeate flux was significantly lower for the G-10 membrane although the G-10 membrane is a loose nanofiltration membrane with a reported 2500 Da MWCO compared to the HL membrane with a reported 300-500 Da MWCO. The total peptide transmission, organic and inorganic content of the fractions for the two feed sources and membrane type were affected differently according to pH and NaCl addition. These results indicate that the two feed sources are of different composition and that nanofiltration is a possible method to fractionate peptides. The bioactivity of the nanofiltration fractions was tested as a nutrient additive to a serum-free media in CHO cells. It was shown that the productivity is not always related to the cell density, as the highest overall specific interferon productivity was achieved for low cell density similar to the hydrolysate free negative control. Furthermore, the retentate fraction of yeast extract separated with the G-10 membrane at a pH of 8 resulted in the highest cell density. According to these results, nanofiltration is a promising method for the enrichment of protein hydrolysates as a supplement for serum in cell culture.

The objective of this work was the investigation of nanofiltration as a potential avenue to fractionate protein hydrolysates and produce protein hydrolysate fractions with stimulating bioactivity for the development of a supplement for a serum-free media. Mammalian cell culture is widely used for the production of therapeutic proteins such as antibodies, interleukins, and vaccines because of the ability of mammalian cells to glycosylate proteins. A complex media with the addition of serum is often required to meet the requirements of the cells. Although serum is a supplement that provides different proteins such as growth factors and hormones, serum has several disadvantages such as high cost, difficulty of downstream processing due to its high protein content and the possibility of microbiological contamination. Protein hydrolysates from plant, animal, or yeast cells contain a complex mixture of peptides and amino acids and have been shown to enhance growth of certain mammalian cell lines cultured in serum-free media. To fractionate peptide mixtures, nanofiltration was investigated in this study. Nanofiltration is a pressure driven membrane separation process based on size and charge. The investigation of pH and NaCl on the filtration performance for two different nanofiltration membranes (HL membrane and G-10 membrane) was achieved using a 24 factorial design. The total peptide concentration, the antioxidant activity, and organic and inorganic content were analyzed in the permeate and retentate fraction. The fractions were also tested for their enhanced growth ability and the specific -interferon productivity with CHO cells. Furthermore the retentate and permeate fractions were analyzed by reversed phase-HPLC to characterize the peptide and free amino acid distribution profile. Through the factorial design, the membrane type was shown to have a significant effect on the filtration performance for both yeast extract and Primatone. A significant difference, but similar for both feed sources, was observed for the total peptide transmission with around 10% for the HL membrane and around 30% for the G-10 membrane. The average permeate flux was significantly lower for the G-10 membrane although the G-10 membrane is a loose nanofiltration membrane with a reported 2500 Da MWCO compared to the HL membrane with a reported 300-500 Da MWCO. The total peptide transmission, organic and inorganic content of the fractions for the two feed sources and membrane type were affected differently according to pH and NaCl addition. These results indicate that the two feed sources are of different composition and that nanofiltration is a possible method to fractionate peptides. The bioactivity of the nanofiltration fractions was tested as a nutrient additive to a serum-free media in CHO cells. It was shown that the productivity is not always related to the cell density, as the highest overall specific interferon productivity was achieved for low cell density similar to the hydrolysate free negative control. Furthermore, the retentate fraction of yeast extract separated with the G-10 membrane at a pH of 8 resulted in the highest cell density. According to these results, nanofiltration is a promising method for the enrichment of protein hydrolysates as a supplement for serum in cell culture.

Cartilage defects have limited capacity for repair and are often replaced by fibrocartilage with inferior mechanical properties. To overcome the limitations of artificial joint replacement, high throughput screens (HTS) could be developed to identify molecules that stimulate differentiation and/or proliferation of articular cartilage for drug therapy or tissue engineering. Currently embryonic stem cells (ESCs) can differentiate into articular cartilage by forming aggregates (embryoid body (EB), pellet, micromass), which are difficult to image. I present a novel, single-step method of generating murine ESC (mESC)-derived chondrocytes in monolayer cultures in chemically defined conditions. Mesoderm induction was achieved in cultures supplemented with BMP4, Activin A or Wnt3a. Prolonged culture with sustained Activin A, TGFβ3 or BMP4 supplementation led to robust chondrogenic induction. A short pulse of Activin A or BMP4 also induced chondrogenesis efficiently while Wnt3a acted as a later inducer. Long-term supplementation with Activin A or with Activin A followed by TGFβ3 may specifically promote articular cartilage formation. Thus, I devised a serum-free (SF) culture system to generate ESC-derived chondrocytes without the establishment of 3D cultures or the aid of cell sorting. Cultures were governed by the same signaling pathways as 3D ESC differentiation systems and limb bud mesenchyme or articular cartilage explant cultures. I am also in the process of creating a Col2a1 promoter-controlled, Cre-inducible reporter cell line to be used in my SF culture system using the Multisite Gateway® cloning technology. ESCs undergoing chondrogenic differentiation can be identified and quantified in HTS via the expression of fluorescent proteins. In addition, this transgenic line can be used to isolate ESC-derived chondrocytes as well as their progeny via cell sorting or antibiotic selection for in-depth characterization. The modular design of my construct system allows transgenic lines to be generated using various promoters of chondrogenic marker genes to perform parallel HTS analyses.

Cartilage defects have limited capacity for repair and are often replaced by fibrocartilage with inferior mechanical properties. To overcome the limitations of artificial joint replacement, high throughput screens (HTS) could be developed to identify molecules that stimulate differentiation and/or proliferation of articular cartilage for drug therapy or tissue engineering. Currently embryonic stem cells (ESCs) can differentiate into articular cartilage by forming aggregates (embryoid body (EB), pellet, micromass), which are difficult to image. I present a novel, single-step method of generating murine ESC (mESC)-derived chondrocytes in monolayer cultures in chemically defined conditions. Mesoderm induction was achieved in cultures supplemented with BMP4, Activin A or Wnt3a. Prolonged culture with sustained Activin A, TGFβ3 or BMP4 supplementation led to robust chondrogenic induction. A short pulse of Activin A or BMP4 also induced chondrogenesis efficiently while Wnt3a acted as a later inducer. Long-term supplementation with Activin A or with Activin A followed by TGFβ3 may specifically promote articular cartilage formation. Thus, I devised a serum-free (SF) culture system to generate ESC-derived chondrocytes without the establishment of 3D cultures or the aid of cell sorting. Cultures were governed by the same signaling pathways as 3D ESC differentiation systems and limb bud mesenchyme or articular cartilage explant cultures. I am also in the process of creating a Col2a1 promoter-controlled, Cre-inducible reporter cell line to be used in my SF culture system using the Multisite Gateway® cloning technology. ESCs undergoing chondrogenic differentiation can be identified and quantified in HTS via the expression of fluorescent proteins. In addition, this transgenic line can be used to isolate ESC-derived chondrocytes as well as their progeny via cell sorting or antibiotic selection for in-depth characterization. The modular design of my construct system allows transgenic lines to be generated using various promoters of chondrogenic marker genes to perform parallel HTS analyses.

Thesis (Ph. D.)--University of Hawaii at Manoa, 1990. / Includes bibliographical references (leaves 68-76) / Microfiche. / xi, 76 leaves, bound ill. 29 cm

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The overall focus of this project has two parts: First, was to develop a protocol utilizing serum-free media formulations and defined plating and culture techniques to create functional in vitro myotubes derived from adult skeletal muscle satellite cells. The second was to manipulate the inherent muscle parameters such as force output and fatigue of these myotubes by employing exercise regimes or by small molecule application. The importance of serum-free medium use for in vitro cultures is becoming increasingly important in creating functional systems that can be validated for drug testing by the Food and Drug Administration (FDA). Also, the study of age related diseases as well as the potential for “personalized medicine” relies on the proliferation and maturation of satellite cells from adult derived tissue. For that purpose, a serum-free medium and culture system was designed to create mature striated myotubes in culture on a defined non-biological substrate N-1[3-trimethoxysilyl propyl] diethylenetriamine (DETA). These myotubes were evaluated by morphology, muscle specific protein expression, and by muscle functionality. After the thorough characterization of the resultant myotubes the functional output of the muscle was altered utilizing chemical means (creatine supplementation and PGC-1? agonists), chronic long term stimulation, and the use of PGC-1? deficient tissue. In this thesis presentation the utility of the newly developed medium formulation to create myotubes from a variety of adult derived muscle sources will be shown. A protocol in which to exercise skeletal muscle in vitro to alter endurance was developed and employed to manipulate skeletal muscle. Finally, small molecules were tested to validate this system for drug study use. This engineered system has the potential for high-throughput screening of drugs for efficacy and drug toxicity studies as well as general biological studies on muscle fatigue.

Global food production management has become a challenge with an anticipated population of 10 billion people by 2050 and the ongoing COVID-19 epidemic. Seafood is a vital food source due to its widespread consumption, excellent nutrient profile, and low feed conversion ratio, rendering its sustainable production quintessential. Cellular agriculture or cultured meat can increase seafood production; however, the conventional use of Fetal Bovine Serum (FBS) in culture media restricts its utilization at an industrial level. FBS is effective but has many limitations: unethical animal extraction, high demand and low supply, poorly defined ingredients, variable performance, and high cost that impedes the feasibility and commercial viability of cellular agriculture. Thus, employing serum-free media becomes a quintessential need for cellular agriculture. This project aims to replace or reduce the typical 10% serum usage in Zebrafish embryonic stem cell (ESC) production media with protein hydrolysates derived from low-cost natural sources with high protein content. Enzymatic hydrolysis was performed on nine sources: insects (black army fly and cricket), plants (pea), fungi (mushroom and yeast), algae, and marine invertebrates (oyster, mussel, and lugworm). The resulting hydrolysates were evaluated for serum replacement in zebrafish ESCs. All hydrolysates were used at five different concentrations (10, 1 0.1, 0.01 and 0.001 mg/mL) in serum concentrations of 10%, 5%, and 0% with four biological replicates. The best hydrolysate sources and concentrations were selected for further testing at 2.5% and 1% serum concentrations. All hydrolysates, except for cricket, could restore or significantly increase cell growth with 50% less serum at a concentration of 0.1-0.001mg/mL. Protein hydrolysate concentration of 10 and 1mg/mL was toxic for cells. Additionally, the eight hydrolysates could reduce serum concentrations up to 75–90%. However, no protein hydrolysate could completely replace serum, as cells using only protein hydrolysates exhibited morphological aberrations and decreased growth. Replacing serum with protein hydrolysates lowers cellular agriculture's overall cost, thus enabling the commercialization of cultured meat and the development of a sustainable food system. In the future, blending various protein hydrolysate sources with or without the addition of conventional growth factors could be done to create the ideal serum-free media. / Doctor of Philosophy / With a predicted population of 10 billion by 2050 and the ongoing COVID-19 outbreak, the management of global food production has become a dilemma. However, due to its widespread consumption and good nutrient profile, seafood is an essential food supply, making its sustainable production indispensable. Both capture fisheries and aquaculture are conventional ways to produce seafood. However, they are under tremendous pressure and require alternatives that can alleviate this demand and contribute to the sustainable growth of seafood. In-vitro cultured meat, also known as lab-grown meat, is a novel technique with the potential to supplement the traditional fish sector. It appears a great option, as it completely imitates meat and offers numerous environmental, financial, and health advantages. A culture medium supports the existence, survival, growth, and multiplication of meat-producing cells and tissues in cell-based meat. However, the culture medium uses a Fetal Bovine Serum (FBS) supplement, which dramatically increases the cost and raises many ethical concerns as it is derived from a cow's fetus. In this thesis, we substitute FBS with protein hydrolysates derived from nine distinct sources. Hydrolysing proteins with enzymes produce protein hydrolysates, rich in nutrients and peptides that promote cell development. Enzymes were used to hydrolyse nine unique and protein-rich sources, including insects (black army fly and cricket), plants (pea), fungi (mushroom and yeast), algae, and marine creatures (oyster, mussel, and lugworm). The resultant hydrolysates were investigated for replacement of serum in cell culture. Eight protein hydrolysates successfully replaced 90% of serum without impairing cell growth and structure but could not replace serum entirely. In the future, serum-free media could be created by combining these various protein hydrolysates with or without adding other growth-promoting components.