Progress 01/01/22 to 12/11/24
Outputs
Target Audience:Decision makers and scientists from academia and foodindustries for novel food ingredients, food innovation,food products, and nutraceutical productsdevelopments would be interested in the outcomes of this cost-effective 3D biomanufacturing technologies for high-efficient production of regenerative animal proteins for human health. The cultured proteins are expected to be highly digestive and an essential amino acid source to secure human nutrients sustainability in long term. Changes/Problems:The Principal Investigator (PI), Dr. Sun, along with her technical staff and graduate students who have been working on this project, has recently relocated to the Wake Forest Institute for Regenerative Medicine (WFIRM) at the Wake Forest University School of Medicine. As a result, the primary research location for this project will be transferred from Kansas State University (KSU) to WFIRM. Dr. Sun will continue to serve as the PI, overseeing the project's success and supervising her team to achieve the proposed objectives. What opportunities for training and professional development has the project provided?Several graduate students and one post-doc research associate were involved in and trained during this project in the area of synthetic and natural protein based hydrogels, biomanufacturing of cell based proteins, 3D cell cultures, conversion of animal blood components to renewable stem cells, regenerative cell based protein process, nutrition attributes, properties measurements, characterizations, statistical analysis, and technical reporting and manuscript development. How have the results been disseminated to communities of interest?Results obtained from this research are mainly disseminated through peer reviewed journal article publications, and or technical conference presentation. What do you plan to do during the next reporting period to accomplish the goals?We have generated substaintial data and will publish 2-4 manuscripts related to cultured animal proteins derived from pig iPSCs in the following years. Objectives for further studies: Conduct scaling up studies at least 1000mL (liter scales level) Investigate plant protein-based hydrogels Evaluate nutritional properties of the cultured pig proteins from pig iPSC both in vitro and in vivo.
Impacts
What was accomplished under these goals?
Tasks investigated in parallel from objectives 1 to 4. Main results are summarized below: Objective 1. Self-renew stem cell reprogramming: In the early stages, in collaboration with our industrial partner, Applied StemCell Inc., we investigated the reprogramming of cattle blood into induced pluripotent stem cells (iPSCs). Lentivirus and Sendai virus vectors were used to reprogram cattle blood collected from local and commercial sources, achieving conversion rates of less than 5%. Literature reports indicated that bovine fibroblast cells demonstrated higher reprogramming efficiency for iPSC generation. Consequently, commercial bovine fibroblast cells were reprogrammed, resulting in improved conversion rates, though they remained insufficient for establishing a robust iPSC line suitable for scalable biomanufacturing studies. The primary challenge was the lack of commercial reprogramming kits utilizing bovine-specific viral vectors. Consequently, we redirected our efforts toward commercially available induced pluripotent stem cells (iPSCs) from meat-producing animals, specifically pigs. Leveraging preliminary work with human iPSCs, we adapted and optimized 3D culture conditions for porcine iPSCs, achieving a robust proliferation rate with a 25- to 35-fold expansion every four days. Compared to the 2D culture of porcine iPSCs on Matrigel, our 3D system delivered 16 times increase in production rate while requiring less space and 8.3 times less culture medium (67,000 cells/ml medium for 2D and 556,000 cells/ml medium for our 3D suspension matrix). Objective 2. Cost effective 3D system for hiPSC culture: Objective 2 Achievement are two parts: Development of a cost-effective scaffolding material: We successfully identified a plant protein-based formula capable of replacing up to 50% of peptide hydrogel without compromising cell growth performance, including growth rate, morphology, and viability. This innovation has the potential to significantly reduce the cost of scaffolding materials used in scalable biomanufacturing. However, the newly identified plant protein composite faces challenges with sterilization methods for large-scale processing, which will be the focus of future investigations. Recyclable cell culture medium: Another major accomplishment was demonstrating that the cell culture medium could be 100% recyclable, further reducing overall production costs. Ten passages of human iPSC preliminarily conducted with a fully recycled medium showed no adverse effects on cell performance, including growth rate, morphology, viability, and gene markers, when compared to controls with 0% recycled medium. The method developed from this study will be adapted for pig iPSCs and further optimized in 2025. Objective 3. Development of scalable 3D hiPSC culture system: Scaling up of 3D culture system has been challenging because of several variables including but not limited to air (Oxygen and CO2 levels) quality and flow rate, stirring rate, cell loading density, culture volume, fresh medium feeding rate, and harvesting schedule. With fixed cell seeding density and air flow level and limited stirring using pipetting, scales from 75 ml to 500 ml was successfully achieved. We have been ready to study the effects of stirring types and stirring rate on cell performance in a 500ml -1000ml bioreactor scale. However, this task has been delayed due to bioreactor installation and training process by the bioreactor's manufacture. We will conduct further scaling up studies. Objective 4. hiPSC crude proteins were used for nutritional attributes analysis: human iPSCs were used as pilot studies in the beginning of this project when animal iPSCs were not available. We found that the crude proteins from human iPSCs possess high value essential amino acid composition than human skeletal muscle protein. With this encouraging data, we will conduct in vitro and in vivo studies using rats' model to evaluate in vivo nutritional values of the RAPs derived from pig iPSCs in the following years when scalable culture conditions for pigs are established.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Cui, Muyun, Wei Wu , Quan Li , Guangyan Qi, Xuming Liu, Jianfa Bai, Mingshun Chen, Ping Li, and Xiuzhi S. Sun*. 2024, Unlocking the Potential of human-Induced Pluripotent Stem Cells: Cellular Responses and Secretome Profiles in Peptide Hydrogel 3D Culture. Cells 2024, 13,143.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Sun, X.S., D. Wang, W. Wang, J. Jaeger, G. Qi, Q. Li, S. Rahaman. 2024. USDA-NIFA 2024 NIFA Novel Foods and Innovative Manufacturing Technologies (A1364). June 17-18, 2024. Amherst , MA.
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Progress 01/01/23 to 12/31/23
Outputs
Target Audience:Decision makers and scientists from academia and foodindustries for novel food ingredients, food innovation,food products, and nutraceutical productsdevelopments would be interested in the outcomes of this cost-effective 3D biomanufacturing technologies for high-efficient production of regenerative animal proteins for human health. The cultured proteins are expected to be highly digestive and an essential amino acid source to secure human nutrients sustainability in long term. The outcomes of this research include cost-effective 3D biomanufacturing technologies for high-efficient production of regenerative animal proteins. The cultured proteins are expected to be highly digestive and an essential amino acid source, and particularly, for those who need energy boost within a few min such as athletes and military soldiers or those who have a sensitive digestive system. Enabling production of high-quality regenerative proteins is a strategic way to secure food sustainability in long term. Changes/Problems:Animal proteins provide all essential amino acids that are involved in human metabolism, immune system, and functional proteins synthesis. The goal of this research is to produce affordable regenerative animal proteins (RAP) by utilizing cattle waste components from abattoirs for high-quality drop-in innovative food proteins utilizing plant protein-based hydrogels. Due to the lack of viral vectors derived bovine, conversion of induced pluripotent stem cells (iPSCs) from cattle waste components has faced grand challenges. Therefore, RAPs from pigs will be investigated as alternatives in the following years. The specific objectives of this project are to 1) establish 3D culture conditions for iPSCs from pigs; 2) develop low-cost scalable plant protein-based hydrogel for applicable three-dimensional (3D) scaffolding for cell expansion and maintenance; 3) identify 3D bioreactor manufacturing and demonstrate RAP production from self-renewable stem cells by physical engineering approaches at kilo gram scale and conduct techno-economic analysis; and 4) assess nutritional quality and protein efficiency and protein digestibility of the RAPs in vitro and in vivo rat model. What opportunities for training and professional development has the project provided?Several graduate students and one post-doc research associate were involved in and trained during this project in the area of synthetic and natural protein based hydrogels, biomanufacturing of cell based proteins, 3D cell cultures, conversion of animal blood components to renewable stem cells, regenerative cell based protein process, nutrition attributes, properties measurements, characterizations, statistical analysis, and technical reporting and manuscript development. How have the results been disseminated to communities of interest?Results obtained from this research are mainly disseminated through peer reviewed journal article publications, and or technical conference presentation. For examples: Xu, Shan1, Guangyan Qi1, Timothy Durrett, Yonghui Li, Xuming Liu, Jianfa Bai, Mingshun Chen, Xiuzhi Susan Sun*, Weiqun Wang*, 2023, High Nutritional Quality of Human induced Pluripotent Stem Cells-generated Proteins through Advanced Scalable Peptide Hydrogel 3D Suspension System, Foods, 12(14), 2713, https://doi.org/10.3390/foods12142713. (1Xu and Qi are equal contribution authors, *corresponding authors). Cui, Muyun, Wei Wu†, Quan Li†, Guangyan Qi, Xuming Liu, Jianfa Bai, Mingshun Chen, Ping Li, and Xiuzhi S. Sun*. 2024, Unlocking the Potential of human-Induced Pluripotent Stem Cells: Cellular Responses and Secretome Profiles in Peptide Hydrogel 3D Culture. Cells 2024, 13,143. https://doi.org/10.3390/cells13020143 Sun, X.S. and Qi, G. 2023, Peptide hydrogel, composition, and conditions for scalable 3D stem cells manufacturing and applications, United States Provisional Patent Application No.: 63/512,820, Sun, X.S., D. Wang, W. Wang, J. Jaeger, G. Qi, Q. Li, S. Rahaman, M. Cui, S. Xu. 2023. USDA-NIFA 2023 NIFA Novel Foods and Innovative Manufacturing Technologies (A1364). June 5-7, 2023. Davis, CA. Sun, X.S. Advanced Synthetic Hydrogels for Life Science Research and Applications. 3rd International Conference on Carbon Chemistry and Materials, October 23-27, 2023, Paris, France. What do you plan to do during the next reporting period to accomplish the goals? Establish iPSCs from pigs for regenerative pig proteins Scale up to at least 1000ml Continue to investigate plant protein based hydrogels Conduct in vivo studies.
Impacts
What was accomplished under these goals?
Several tasks are under investigation in parallel from objectives 1 to 4. Main results are summarized below: Objective 1. Self-renew stem cell reprogramming: In 2022 and 2023, in collaboration with industrial collaborator (Applied StemCell inc.), we investigated conversion of cattle blood into iPSCs. Lentivirus and Sendai virus were used, respectively, to reprogram cattle blood collected from local and commercial sources into iPSCs and conversion rates were not higher than 5%. According to literature, bovine fibroblast cells showed higher conversion rate for iPSCs and thus, commercial bovine fibroblast cells were reprogramed, and conversion rate was improved but still not good enough for establishing a healthy iPSC line to work with. The main reason was due to the lack of commercial reprogramming kit with bovine viral vectors. Therefore, commercial iPSCs derived from meat animal pigs will be investigated in year 2024. 3D culture conditions used for human iPSCs will be adapted and established with modification for iPSCs from pigs. Objective 2. Cost effective 3D system for hiPSC culture: Since there are no cattle iPSCs available yet in 2023, human iPSCs were used for objectives 2 and 3. In objective 2, we have investigated to replace portion of peptide hydrogel with low-cost plant-based proteins without trading off cell growth performance and integrity at gene marker levels. We have identified a plant protein-based formula to replace up to 50% of peptide hydrogel without altering cell growth performance in terms of cell growth rate, morphology, and viability. Gene markers mainly focusing on pluripotent markers were characterized by RT-qPCR and results showed hiPSCs gene markers at 50% replacement remained similar as control samples with 0% replacement. However, the hurdle of using this newly identified plant based protein composite for 3D hiPSC culture is the sterilization method of the plant protein, which will be further determined. Another major accomplishment in objective 2 is that the cell culture medium can be 100% recyclable, which should further reduce the overall production cost. Ten passages were conducted using 100% cultured medium recycle showed that cell performance (cell growth rate, morphology, and viability) remained similar as to control with 0% cultured medium recycle. Pluripotent gene markers (characterized by RT-qPCR) of hiPSCs over ten passages remained similar as control samples with 0% cultured medium recycle. Objective 3. Development of scalable 3D hiPSC culture system: Scaling up of 3D culture system has been challenging because of several variables including but not limited to air (Oxygen and CO2 levels) quality and flow rate, stirring rate, cell loading density, culture volume, fresh medium feeding rate, and harvesting schedule. With fixed cell seeding density and air flow level and limited stirring using pipetting, scales from 75 ml to 500 ml was successfully achieved in 2023. We are ready to study the effects of stirring types and stirring rate on cell performance in a 500ml -1000ml bioreactor scale in 2024. Objective 4. hiPSC crude proteins were used for nutritional attributes analysis: The crude RAPs from hiPSCs were found to possess high value essential amino acid composition than human skeletal muscle protein, which should be valuable nutraceutical ingredients to improve human health. We will conduct in vivo studies using rats' model to evaluate in vivo nutritional values of the RAPs in the following years when scalable culture conditions for pigs are established.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Xu, Shan1, Guangyan Qi1, Timothy Durrett, Yonghui Li, Xuming Liu, Jianfa Bai, Mingshun Chen, Xiuzhi Susan Sun*, Weiqun Wang*, 2023, High Nutritional Quality of Human induced Pluripotent Stem Cells-generated Proteins through Advanced Scalable Peptide Hydrogel 3D Suspension System, Foods, 12(14), 2713, https://doi.org/10.3390/foods12142713. (1. Xu and Qi are equal contribution authors, *corresponding authors).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Sun, X.S., D. Wang, W. Wang, J. Jaeger, G. Qi, Q. Li, S. Rahaman, M. Cui, S. Xu. 2023. USDA-NIFA 2023 NIFA Novel Foods and Innovative Manufacturing Technologies (A1364). June 5-7, 2023. Davis, CA.
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Progress 01/01/22 to 12/31/22
Outputs
Target Audience:Decision makers and scientists from academia and foodindustries for noval food ingredients, food innovation,food products, neutroceutical productsdevelopments would be interested in the outcomes of this research realted tocost-effective 3D biomanufacturing technologies for high-efficient production of regenerative animal proteins for food applications. The cultured proteins are expected to be highly digestive and an essential amino acid source for everyone, and particularly, for those who need energy boost within a few min such as athletes and military soldiers or those who have a sensitive digestive system. Producing high-quality animal proteins from animal blood waste would increase animal life cycle that benefits the animal production system and meat industries, as well as support food sustainability. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Several graduate students and one post-doc research associate were involved in and trained during this project in the area of synthetic and natural protein based hydrogels, biomanufacturing of cells based proteins, 3D cell cultures, conversion of blood components to renewable stem cells, regenerative cell based protein process, nutrition attributes, properties measurements, characterizations, statistical analysis, and technical reporting and manuscript development. How have the results been disseminated to communities of interest?Results obtained from this research are mainly disseminated through peer reviewed journal article publications, and or technical conference presentation. One peer reviewed article has been submitted to Foods and is under review. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Several tasks are under investigation in parallel from objectives 1 to 4. Main results are summarized below: Objective 1. Bovine iPSC reprogramming: We have obtained skillful methods for cattle blood samples processing and handling into mycoplasma samples. These mycoplasma samples were sent to our industrial collaborator (Applied Stemcell Inc) for iPSC reprogramming. Since there has been no bovine viral vectors available, Lentivirus kit for mouse iPSC reprogramming was used because, according to literature, mouse viral vectors showed positive results for reprogramming bovine fibroblast cells into iPSCs. Three trials were conducted using Lentivirus kit, however, the conversion was not successful due to the lack of bovine viral vectors for iPSC reprogramming. Advanced Sendai reprogramming kit is currently under investigation with the hope to improve bovine blood reprogramming. ?Objective 2. Plant protein-based hydrogel for 3D iPSC culture: human iPSC (hiPSC) was used for the task of developing plant protein-based hydrogel since cattle iPSCs is under development in parallel. Once the cattle iPSCs is achieved, it would be easier to adapt the methods developed with hiPSCs to cattle iPSCs. Major accomplishments in objective 2 are summarized below: Two types of natural proteins have been investigated. By replacing peptide hydrogel with varied plant based proteins from 0% to 100% for 3D hiPSC cultures, 50% replacement has been identified to produce hiPSCs without altering the cell performance in terms of cell viability, proliferation, and morphology. Cell culture medium (CCM) is another expensive factor for regenerative protein productions. Therefore, the feasibility of used CCM recycling has been investigated to further reduce production cost. Three different treatments were examined including a) cell metabolic wastes (CMW) was removed from the used CCM; b) the CMW was not removed but the CCM was filtered with 0.22 µm filter to remove some particles possibly derived from scaffolding hydrogel, or dead cell debris, and or possible bacteria and mold; and c) 100% used CCM was recycled by adding 10% of the CCM into fresh CCM for next passage. Results showed that all three treatments had similar cell performance as the fresh CCM. Therefore, with proliferation rate of 10 folds or higher, 100% used CCM can be recycled for next generation iPSC culture. Ten passages have been achieved, and consistent cell proliferation, viability and morphology were obtained. Cell integrity at gene marker level of the hiPSCs cultured in plant protein-based hydrogel and recycled medium are under characterization. Objective 3. Development of scalable 3D iPSC culture system: Scaling up of 3D culture system has been challenging because of several variables including but not limited to air (Oxygen and CO2 lelvels) quality and flow rate, stirring rate, cell loading density, culture volume, fresh medium feeding rate, and harvesting schedule. With fixed cell seeding density and air flow level and limited stirring using pipetting, scales from 0.5 ml to 10 ml was successfully achieved in early of the year, and then 75 ml scale processing has recently been achieved with satisfied cell performance. We are aiming to study the effects of stirring types and stirring rate on cell performance in a 1000ml bioreactor scales. Objective 4. hiPSC crude proteins were used for nutritional attributes analysis. The amino acid composition of hiPSC analyzed by hydrophilic interaction chromatography coupled with tandem mass spectrometry (HILIC-MS/MS) show that the hiPSCs-generated proteins demonstrated a significant higher essential amino acid (EAA) content (39.0%) than human skeletal muscle protein (31.8%). Leucine content was the highest among the EAA tested, which is a key factor translating diet quality into the muscle protein synthesis response to meals. In vitro protein digestibility of hiPSCs-generated proteins was evaluated by pH-drop method, and showed a significant higher (78.0 ± 0.7%) than the commercial beef protein isolate (75.7 ± 0.6%). IACUC protocol for in vivo animal usage has been approved and ready for nutritional assessment.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2023
Citation:
1. Shan Xu1, Guangyan Qi1, Timothy Durrett, Yonghui Li, Xuming Liu, Jianfa Bai, Mingshun Chen, Xiuzhi Susan Sun*, Weiqun Wang*, 2023, High Nutritional Quality of Human induced Pluripotent Stem Cells-generated Proteins through Advanced Scalable Peptide Hydrogel 3D Suspension System, Foods (under review)
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