Progress 09/01/05 to 08/31/10
Outputs
OUTPUTS: While significant advances have been made in numerous aspects of bovine embryo production, little progress has been seen in the ability to select embryos with the greatest developmental capacity and implantation potential. Enhanced selection of bovine embryos with "normal development" and the greatest potential to establish a pregnancy would have an enormous impact on basic and applied science applications, as well as efficiency of offspring production following embryo production and transfer. This proposal presented a design-directed project to develop a novel embryo culture system that will culture multiple single embryos under simulated physiological conditions while simultaneously performing real-time monitoring on biochemical markers of embryo quality. This grant supported the design and fabrication of a biomimetic microbioreactor with computer-controlled valves and pumps to perform integrated embryo culture and biomarker analysis. Three experiments were performed. 1) Demonstrate the ability to quantitate and differentiate lactate production from in vivo- derived and IVP embryos with an in-chip enzymatic assay. 2) Quantitate and compare IGF-I production from developmentally divergent IVP embryos at the 2-cell, 8-cell, morula, and blastocyst stages with microfluidic in-line immunological assays. 3) Integrate microfluidics and real-time capillary electrophoresis for quantification and comparison of the bovine Ped gene product in developmentally divergent IVP embryos at the 2-cell, 8-cell, morula, and blastocyst stages. Dissemination of outputs were presented in abstract, poster and oral presentations at national meetings, placed on our website, published in peer-reviewed journals, and published in reviews and books. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We have demonstrated that microfluidic dynamic culture of immature oocytes significantly improved embryonic developmental competence. Cumulus oocyte complexes (COC) were cultured for 22hrs in microfluidic chip funnels with and without Braille pin-driven media movement. While we have seen no difference in nuclear maturation, fertilization, or first cleavage, we do see a significant enhancement in total blastocyst development. We have recently developed a computerized microfluidic embryo culture and assay device which can perform automated periodic analysis of embryo metabolism over 24 hrs. This system was able to measure glucose consumption of single or multiple (10) live embryos at the blastocyst stage. Approximate glucose consumption of embryos from the multiple and single embryos were 5pmol/hr per embryo and 13pmol/hr per embryo, respectively. These works, in conjunction with our continued work on real-time lactate production will allow us to measure real-time glycolytic rate of embryos on chip. We worked on IGF-1 capillary electrophoresis assay development. This has proved most difficult. We also worked on QA-2 analysis - however the antibody available was non-reliable, non-specific, and not useful for a capillary electrophoresis assay. Two manuscripts are in preparation in relation to 1) microfluidic bovine embryo culture, and 2) engineering and integration of a single embryo culture and metabolic analysis assessment. In relation to impact, cattle embryos are used in both basic research and for applied purposes. From a research focus, embryo production is of great importance in understanding events in oocyte developmental competence, extracellular and intracellular events important for fertilization, molecular control of embryo development and metabolism, and epigenetic regulation of genetic imprinting and normal development. As an applied technology embryo production provides opportunities to increase number of offspring from superior genotypes. Cloning technologies coupled with embryo production offer future possibility of taking replication of superior individuals to the next level. Lastly, advancements in molecular genetics, cloning, and embryo production/screening provides an opportunity to produce offspring with targeted enhanced or reduced autologous gene expression or inserted inducible heterologous gene products. While significant advances have been made in numerous aspects of bovine embryo production, little progress has been seen in the ability to select embryos with the greatest developmental and implantation potential. Enhanced selection of bovine embryos with "normal development" and the greatest potential to establish a pregnancy would have an enormous impact on all of the above-mentioned basic and applied science applications. This proposal presents a design-directed project to develop a novel microfluidic bioreactor that will culture multiple single embryos under simulated physiological conditions while simultaneously performing real-time monitoring on biochemical markers of embryo quality.
Publications
- Smith GD, Bormann C, Takayama S. Microfluidic Embryo Culture. In: Practical Manual of In Vitro Fertilization: Advanced Methods. Eds. A Agarwal, A Varghese and ZP Nagy. Humana Press (2010) accepted.
- Bormann C, Wheeler M, Beebe D, Takayama S, Smith GD. Microfluidics for In Vitro Fertilization. In: Micro- and Nanoengineering of the Cell Microenvironment: Technologies and Applications. Eds. A Khademhosseini, J Borenstein, S Takayama, M Toner. Artech House Publishing Inc. (2008) 209-225.
- Swain JE, Pool, TB, Takayama S, Smith GD. Microfluidics in ART: Current Progress and Future Directions. In: Textbook of Assisted Reproductive Techniques Laboratory and Clinical Perspectives, Third Edition. Eds. D Gardner, A Weissman, C Howles, Z Shoham. Informa Healthcare Ltd (2009) 843-858.
- Heo YS, Cabrera LM, Bormann C, Shah CT, Takayama S, Smith GD. Dynamic microfunnel culture enhances embryo development and pregnancy rates. Human Reprod (2010; Cover Article) 25:613-622.
- Smith GD, Swain JE, and Bormann, CL. Microfluidics for gametes, embryos, and embryonic stem cells. Seminars in Reproductive Medicine (2010) 29:5-14.
- Swain, JE and Smith GD. Advances in embryo culture platforms: novel approaches to improve preimplantation embryo development through modifications of the microenvironment. Human Reproduction Update 2011, In press
- Bormann CL, Cabrera LM, Heo YS, Kato N, Takayama S, Smith GD. Dynamic culture with microfludic biomemetic conditions improved bovine oocyte and embryo developmental competence. Reproduction (2010) submitted, in revision.
- Heo YS, Cabrera LM, Bormann CL, Shah CL, Tung YC, Smith GD, Takayama S. Real time culture and analysis of single embryo metabolism using a microfluidic device with deformation-based actuation. 2011 In preparation.
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Progress 09/01/06 to 08/31/07
Outputs
We have demonstrated that microfluidic dynamic culture of immature oocytes significantly improved embryonic developmental competence. In the first experiment we placed 10 bovine COCs per 50 microliters TCM-199, supplemented with 10% FCS, bFSH, and EGF, overlaid with oil, at 39C in 5%CO2 and air. Cumulus oocyte complexes (COC) were cultured for 22hrs in microfluidic chip funnels with and without Braille pin-driven media movement. Nuclear maturation to MII was similar between the treatment groups (static - 89%, n=40; dynamic - 91%, n=47). In the second experiments we repeated this experimental design for dynamic versus static maturation, however after 22hrs of maturation all COCs were transferred to 50 microliter microdrops and co-incubated with bovine sperm at 1X106/ml in IVF-TALP for 12h at 39C in 5% CO2. Presumptive zygotes were transferred to 50 microliter microdrops of KSOM + 3mg/ml BSA in 5% CO2, 5% O2, and balanced N2 and cultured for 144hrs at 39C. While we have
seen no difference in nuclear maturation, fertilization, or first cleavage, we do see a significant enhancement in total blastocyst development. We also see a non-significant trend toward more advanced blastocyst development in oocytes matured in microfluidic dynamic versus static culture. We continue our work on the development of on-chip metabolism assays. We have recently developed a computerized microfluidic embryo culture and assay device which can perform automated periodic analysis of embryo metabolism over 24 hrs. The computer-controlled Braille display fluid actuation scheme was based on a design described previously. We used a modified "gated injection" scheme to perform sample injection, reagent mixing, enzyme reaction (30 min) incubation, and sample detection. Samples (or reference) and reagents were introduced from three reservoirs into a reaction channel using a pin actuation sequence for enhanced mixing. After 30 min incubation, washing buffer pushed the sample through
a detection zone where fluorescence intensity was measured, and finally out to the buffer waste reservoir. An automated program used this gated injection scheme to sequentially measure fluorescence from sample, reference, and background every hour for 24 hours. Two different samples containing 5) microM and 100 microM glucose were loaded and their intensities were measured serially. The low standard deviation over 24hr demonstrated the accuracy and stability of this microfluidic analysis system. Furthermore, this system was able to measure glucose consumption of single or multiple (10) live embryos at the blastocyst stage. Approximate glucose consumption of embryos from the multiple and single embryos were 5pmol/hr per embryo and 13pmol/hr per embryo, respectively. These works, in conjunction with our continued work on real-time lactate production will allow us to measure real-time glycolytic rate of embryos on chip. Work continues on IGF-1 capillary electrophoresis assay development.
This has proved most difficult. We also continue work on QA-2 analysis.
Impacts
Cattle embryos are used in both basic research and for applied purposes. From a research focus, embryo production is of great importance in understanding events in oocyte developmental competence, extracellular and intracellular events important for fertilization, molecular control of embryo development and metabolism, and epigenetic regulation of genetic imprinting and normal development. As an applied technology embryo production provides opportunities to increase number of offspring from superior genotypes. Cloning technologies coupled with embryo production offer future possibility of taking replication of superior individuals to the next level. Lastly, advancements in molecular genetics, cloning, and embryo production/screening provides an opportunity to produce offspring with targeted enhanced or reduced autologous gene expression or inserted inducible heterologous gene products. While significant advances have been made in numerous aspects of bovine embryo
production, little progress has been seen in the ability to select embryos with the greatest developmental and implantation potential. Enhanced selection of bovine embryos with "normal development" and the greatest potential to establish a pregnancy would have an enormous impact on all of the above-mentioned basic and applied science applications. This proposal presents a design-directed project to develop a novel microfluidic bioreactor that will culture multiple single embryos under simulated physiological conditions while simultaneously performing real-time monitoring on biochemical markers of embryo quality.
Publications
- Heo, Y., L. Cabrera, J. Song, N. Futai, Y. Tung, G. Smith, and S. Takayama. 2007. Characterization and resolution of evaporation-mediated osmolality shifts that constrain microfluidic cell culture in poly(dimethylsiloxane) devices. Anal Chem 79:1126-1134.
- Smith, G.D., and Takayama, S. 2007. Gamete and embryo isolation and culture with microfluidics. Therio. 68:S190-S195.
- Bormann, C.L., Wheeler, M., Beebe, D., Takayama, S., Smith, G.D. 2007. Microfluidics for In Vitro Fertilization. In: Micro- and Nanoengineering of the Cell Microenvironment: Techniques and Applications. In Press.
- Swain, J.E., Pool, T., Takayama, S., Smith, G.D. 2007. Microfluidics in ART. In: Textbook of Assisted Reproductive Techniques: Laboratory and Clinical Perspectives. In Press. Bormann, C., L. Cabrera, Y. Heo, S. Takayama, and G. Smith. 2007. Dynamic microfluidic embryo culture enhances blastocyst development of murine and bovine embryos, SSR.
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Progress 09/01/05 to 09/01/06
Outputs
A microfluidic embryo culture system that utilized Braille-pin actuation to produce dynamic fluid was developed with mouse embryos where we get significantly more advanced embryo development. IVP bovine zygotes have been tested in microfluidic dynamic culture versus control microfluidic chips with no fluid movement (static). With dynamic culture (n=97 zygotes) 64.9% cleaved, 35% developed to blastocyst, yielding a 54% cleavage to blastocyst conversion rate. With static culture (n=145 zygotes) 66.2% cleaved, 21.4% developed to blastocyst, yielding a 32.3% cleavage to blastocyst conversion rate. These experiments were generated from pooled zygotes, with the same sequential culture media, under the same gaseous and environmental culture conditions - except for the dynamic versus static fluid environment. We have developed an assay system to measure glucose and lactate with a high sensitivity, specificity, and without use of ultraviolet light. In this system (example
lactate measure) lactate oxidase reacts with L-lactate to form pyruvate and H2O2. In the presence of horseradish peroxidase (HRP), H2O2 reacts with Amplex UltraRed to generate a red-fluorescent oxidation product, Resorufin. After assay optimization we measured individual embryo lactate production at the 2-cell (n=22) and blastocyst stage (n=25). Average lactate production/hour for individual 2-cell embryos is 7.47+1.09 and blastocyst is 14.38+1.76 picomoles. Second we fabricated of microfluidic chip for measurement of embryos metabolism with fluid actuation, valving, pumping and mixing. Chip design has loading channels that merge into a short, conversing channel, which is connected to the middle of a reaction channel. Reaction channels have a buffer reservoir at the beginning and branches off two directions at the end; one for waste and the other for detection. Reagents are introduced and a push/pull mixing sequence is employed. We have also successfully performed initial quantitative
testing of glucose and lactate assay capabilities and can successfully measure serial glucose concentrations over 6 hour on chip. Using Electron-spray and MALDI-TOF proteomics we have identified 43 novel embryo secreted proteins for future biomarker analysis. CE immunoassays have focused on IGF-1. Development of a noncompetitive assay has been unsuccessful, likely because addition of the relatively small IGF-I (7 kDa) to the antibody-Protein G complex (183 kDa) does not alter electrophoretic mobility of the complex sufficiently to allow resolution. We are investigating higher resolution separation conditions. We have also investigated competitive CE immunoassay where it is necessary to fluorescently label IGF-I. Carboxyfluorescein succinimidyl ester (CFSE) has been shown to directly label lysine residues and terminal amines on the IGF-I protein. We observe 2 peaks for labeled IGF-I. We hypothesize that these peaks correspond to different numbers of CFSE attached to the IGF-I. Further
work is planned to optimize the dye-to-protein ratio and remove excess CFSE from the IGF* solution. The next step will be to purify the labeled IGF-I and determine the separation conditions for the competitive assay.
Impacts
Cattle embryos are used in both basic research and for applied purposes. From a research focus, embryo production is of great importance in understanding events in oocyte developmental competence, extracellular and intracellular events important for fertilization, molecular control of embryo development and metabolism, and epigenetic regulation of genetic imprinting and normal development. As an applied technology embryo production provides opportunities to increase number of offspring from superior genotypes. Cloning technologies coupled with embryo production offer future possibility of taking replication of superior individuals to the next level. Lastly, advancements in molecular genetics, cloning, and embryo production/screening provides an opportunity to produce offspring with targeted enhanced or reduced autologous gene expression or inserted inducible heterologous gene products. While significant advances have been made in numerous aspects of bovine embryo
production, little progress has been seen in the ability to select embryos with the greatest developmental and implantation potential. Enhanced selection of bovine embryos with "normal development" and the greatest potential to establish a pregnancy would have an enormous impact on all of the above-mentioned basic and applied science applications. This proposal presents a design-directed project to develop a novel microfluidic bioreactor that will culture multiple single embryos under simulated physiological conditions while simultaneously performing real-time monitoring on biochemical markers of embryo quality.
Publications
- Bormann CL, Cabrera LM, Chisolm C, Heo YS, Takayama S, Smith GD. Development of a sensitive assay to measure lactate production of preimplantation embryos. ASRM 2006 abstract, oral presentation.
- Bormann CL, Swain JE, Ding J, Qihui N, Kennedy RT, Smith GD. Preimplantation embryo secretome identification. ASRM 2006 abstract, oral presentation.
- Cabrera, LM, Heo YS, Takayama S, Smith GD. Improved blastocyst development with microfluidics and braille pin actuator enabled dynamic culture. ASRM 2006 abstract, oral presentation, prize paper candidate.
- Bormann C, Wheeler M, Beebe D, Takayama S, Smith GD. Microfluidics for In Vitro Fertilization. In: Micro- and Nanoengineering of the Cell Microenvironment: Technologies and Applications. Eds. A Khademhosseini, J Borenstein, S Takayama, M Toner. Artech House Publishing Inc. (2007) in preparation.
- Smith GD and Takayama S. Microfluidics in ART. In: Textbook of Assisted Reproductive Techniques Laboratory and Clinical Perspectives, Third Edition. Eds. D Gardner, A Weissman, C Howles, Z Shoham. Informa Healthcare Ltd (2007) in preparation.
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