Developing experimental design and troubleshooting skills in an advanced biochemistry lab

Two skills critically important to all scientists are the ability to design good experiments and to troubleshoot experiments that do not yield the expected results. In spite of their importance, however, these skills are rarely taught as a part of the undergraduate science curriculum. This deficiency was addressed by creating an advanced biochemistry laboratory course that focuses on the development of experimental design and troubleshooting skills. The course provides students with the opportunity to design and evaluate their own experiments through semester-long independent projects that have a common theme (in this case, protein purification, but any theme could be used). Students plan their projects and carry through the experiments by consulting the primary research literature. The instructor provides minimal input in troubleshooting situations, instead allowing the student to think through and implement alternative solutions. A 2-year survey of the course revealed that students were often frustrated but felt the course significantly improved their experimental laboratory skills as well as introducing them to “real-world” laboratory experience. They further indicated that they preferred the approach used in this course to directed “cook-book” laboratory experiences.     

不同培养基对PGPR实验效果的影响

通过对我院生物工程专业本科生的2项PGPR实验,对不同培养基对实验效果的影响进行了初步研究。实验证明,采用不同的培养基,对实验效率和实验效果影响很大;科学合理的选择培养基,能够提高实验结果的准确性和可靠性,可以大幅度地缩短实验时间,提高实验效率,通过本实验的进行达到了引导学生开阔思路,科学合理地设计试验方案,提高综合实验能力的目的。     

Virtual parameter-estimation experiments in Bioprocess-Engineering education

Cell growth kinetics and reactor concepts constitute essential knowledge for Bioprocess-Engineering students. Traditional learning of these concepts is supported by lectures, tutorials, and practicals: ICT offers opportunities for improvement. A virtual-experiment environment was developed that supports both model-related and experimenting-related learning objectives. Students have to design experiments to estimate model parameters: they choose initial conditions and ‘measure’ output variables. The results contain experimental error, which is an important constraint for experimental design. Students learn from these results and use the new knowledge to re-design their experiment. Within a couple of hours, students design and run many experiments that would take weeks in reality. Usage was evaluated in two courses with questionnaires and in the final exam. The faculties involved in the two courses are convinced that the experiment environment supports essential learning objectives well.     

Apoptosis: a four-week laboratory investigation for advanced molecular and cellular biology students

Over the past decade, apoptosis has emerged as an important field of study central to ongoing research in many diverse fields, from developmental biology to cancer research. Apoptosis proceeds by a highly coordinated series of events that includes enzyme activation, DNA fragmentation, and alterations in plasma membrane permeability. The detection of each of these phenotypic changes is accessible to advanced undergraduate cell and molecular biology students. We describe a 4-week laboratory sequence that integrates cell culture, fluorescence microscopy, DNA isolation and analysis, and western blotting (immunoblotting) to follow apoptosis in cultured human cells. Students working in teams chemically induce apoptosis, and harvest, process, and analyze cells, using their data to determine the order of events during apoptosis. We, as instructors, expose the students to an environment closely simulating what they would encounter in an active cell or molecular biology research laboratory by having students coordinate and perform multiple tasks simultaneously and by having them experience experimental design using current literature, data interpretation, and analysis to answer a single question. Students are assessed by examination of laboratory notebooks for completeness of experimental protocols and analysis of results and for completion of an assignment that includes questions pertaining to data interpretation and apoptosis.     

Leading Students to Investigate Diffusion as a Model of Brine Shrimp Movement

Integrating experimental biology laboratory exercises with mathematical modeling can be an effective tool to enhance mathematical relevance for biologists and to emphasize biological realism for mathematicians. This paper describes a lab project designed for and tested in an undergraduate biomathematics course. In the lab, students follow and track the paths of individual brine shrimp confined in shallow salt water in a Petri dish. Students investigate the question, “Is the movement well characterized as a 2-dimensional random walk?” Through open, but directed discussions, students derive the corresponding partial differential equation, gain an understanding of the solution behavior, and model brine shrimp dispersal under the experimental conditions developed in class. Students use data they collect to estimate a diffusion coefficient, and perform additional experiments of their own design tracking shrimp migration for model validation. We present our teaching philosophy, lecture notes, instructional and lab procedures, and the results of our class-tested experiments so that others can implement this exercise in their classes. Our own experience has led us to appreciate the pedagogical value of allowing students and faculty to grapple with open-ended questions, imperfect data, and the various issues of modeling biological phenomena.     

Comparing programmed cell death in cultured primary and tumor cells in inquiry-based cell biology laboratory exercises

ABSTRACT

Programmed cell death, or apoptosis, is a cellular pathway by which individual cells self-destruct for the benefit of the organism. In this practical paper, we describe laboratory exercises with an inquiry-based learning (IBL) approach in which undergraduate students compared apoptosis among different types of cultured cells. Ultraviolet (UV) radiation was used to induce apoptosis in mouse primary cells and in Chinese hamster ovary (CHO) tumor cells. Students hypothesized that, because tumor cells evade apoptosis, CHO cells would exhibit less apoptosis compared to primary cells. Treated and control cells were labeled for two hallmarks of apoptosis, fragmented DNA and active caspase-3 enzyme, and all nuclei were visualized with DAPI. Cell counts were conducted using fluorescence microscopy. Exposure to UV light induced apoptosis in both cell types compared to controls, but no significant difference in the proportions of labeled cells was observed between UV-treated primary and CHO cells. Optimal parameters for UV exposure and labeling techniques are presented. This exercise provides instructors with methodology for allowing students to use a basic cell culture system and microscopy to formulate a hypothesis and design experiments related to apoptosis. Students incorporated their work into a research paper, which served as the main mode of assessment.     

Biotechnology apprenticeship for secondary-level students: teaching advanced cell culture techniques for research

The purpose of this article is to discuss small-group apprenticeships (SGAs) as a method to instruct cell culture techniques to high school participants. The study aimed to teach cell culture practices and to introduce advanced imaging techniques to solve various biomedical engineering problems. Participants designed and completed experiments using both flow cytometry and laser scanning cytometry during the 1-month summer apprenticeship. In addition to effectively and efficiently teaching cell biology laboratory techniques, this course design provided an opportunity for research training, career exploration, and mentoring. Students participated in active research projects, working with a skilled interdisciplinary team of researchers in a large research institution with access to state-of-the-art instrumentation. The instructors, composed of graduate students, laboratory managers, and principal investigators, worked well together to present a real and worthwhile research experience. The students enjoyed learning cell culture techniques while contributing to active research projects. The institution's researchers were equally enthusiastic to instruct and serve as mentors. In this article, we clarify and illuminate the value of small-group laboratory apprenticeships to the institution and the students by presenting the results and experiences of seven middle and high school participants and their instructors.     

Using the scratch assay to study cell migration in an inquiry-based cell biology lab

Cell migration, a fundamental process in development, wound healing, and immune function, is a common topic in undergraduate cell biology courses. We developed laboratory exercises with an inquiry-based learning (IBL) approach in which cell migration could be examined with the scratch assay, adapted from the primary literature. A narrow scratch was created in a confluent monolayer of cells growing on the bottom of a cell culture dish. Migration into the resultant cell-free zone from both sides of the scratch was measured after one day using the scale bar function of a digital camera. The Chinese hamster ovary cell line was used, but any adherent cell type could be examined. Students used the scratch assay to formulate hypotheses and design experiments in which variables affecting cell migration could be investigated. For example, the effect of cytoskeletal disruption was evaluated by adding the microtubule- and microfilament-disrupting drugs, colcemid and phallacidin, respectively, to the growth medium when the scratch was made. Optimal drug concentration parameters were determined for students to reference. Low drug concentrations inhibited cell migration, while higher concentrations killed the cells. This study demonstrated that the scratch assay is an accessible IBL method for studying cell migration.     

植物系统分类学实验教学改革实践

植物学是一门实践性非常强的学科。系统分类部分是植物学实验教学中的重要内容。针对传统植物系统分类学实验教学中的弊端,采取以下6点措施开展教学改革实践：1）将实验教学与理论教学有机结合;2）要求学生参与实验材料准备,调动学生主观能动性;3）开展小组学习,发挥学生主体作用;4）以兴趣促学习,拓展教学内容;5）开设创新性实验,培养实践精神;6）科研与教学相结合,提升学生科研能力。     

Selective use of the primary literature transforms the classroom into a virtual laboratory

CREATE (consider, read, elucidate hypotheses, analyze and interpret the data, and think of the next experiment) is a new method for teaching science and the nature of science through primary literature. CREATE uses a unique combination of novel pedagogical tools to guide undergraduates through analysis of journal articles, highlighting the evolution of scientific ideas by focusing on a module of four articles from the same laboratory. Students become fluent in the universal language of data analysis as they decipher the figures, interpret the findings, and propose and defend further experiments to test their own hypotheses about the system under study. At the end of the course students gain insight into the individual experiences of article authors by reading authors' responses to an e-mail questionnaire generated by CREATE students. Assessment data indicate that CREATE students gain in ability to read and critically analyze scientific data, as well as in their understanding of, and interest in, research and researchers. The CREATE approach demystifies the process of reading a scientific article and at the same time humanizes scientists. The positive response of students to this method suggests that it could make a significant contribution to retaining undergraduates as science majors.     

Application of process quality engineering techniques to improve the understanding of the in vitro processing of stem cells for therapeutic use

The translation of experimental cell-based therapies to volume produced commercially successful clinical products requires the development of capable, economic, scaleable (and therefore frequently necessarily automated) manufacturing processes. Application of proven quality engineering techniques will be required to interrogate, optimise, and control in vitro cell culture processes to regulatory and clinically acceptable specifications. We have used a Six Sigma inspired quality engineering approach to design and conduct a factorial screening experiment to investigate the expansion process of a population of primary bone marrow-derived human mesenchymal stem cells on a scaleable automated cell culture platform. Key cell culture process inputs (seeding density, serum concentration, media quantity and incubation time) and important cell culture process responses (cell number and the expression of alkaline phosphatase, STRO-1, CD105 and CD71) were identified as experimental variables. The results rank the culture factors and significant culture factor interactions by the magnitude of their effect on each of the process responses. This level of information is not available from conventional single factor cell culture studies but is essential to efficiently identify sources of variation and foci for further process optimisation. Systematic quality engineering approaches such as those described here will be essential for the design of regulated cell therapy manufacturing processes because of their focus on identifying the sources of and the control of variation, an issue that is at the core of current Good Manufacturing Practice.     

Production of xylitol from raw wood hydrolysates by Debaryomyces hansenii NRRL Y-7426

Xylose solutions were obtained from the hemicellulosic fraction of Eucalyptus globulus by means of sulphuric acid treatments performed under selected operational conditions. The hydrolysates were neutralized, supplemented with nutrients, sterilized and utilised as fermentation media for xylitol production using the yeast Debaryomyces hansenii NRRL Y-7426. Preliminary experiments allowed the adaptation of the strain to the culture media. Further fermentation trials were performed with adapted cells according to an incomplete, factorial design of experiments. Empirical models describing the bioconversion were derived from the experimental results. The effects of three operational variables (nutrient concentration, initial pH and shaking speed) on both xylose consumption and xylitol production rates were described by significant mathematical equations. Additional aspects in relation to the process were also discussed. The authors are grateful to Xunta de Galicia and “Orember” for their financial support to this work; as well as to Ms. Rocío Rodríguez Fontán for her excellent technical assistance.     

微生物学检验实验教学的改革与实践

改革传统的微生物学检验实验教学,组织学生到大型医院和医学检验类企业开展实验。按照微生物检验室工作流程,分接种、培养、鉴定3大项目进行实验教学。同时,让学生与企业员工进行深入交流,体验企业文化氛围,感受人文关怀,提高了实验教学质量,让学生树立更科学合理的就业观和择业观。     

Laboratory demonstration of vascular smooth muscle function using rat aortic ring segments

This laboratory exercise uses a simple preparation and a straightforward protocol to illustrate many of the basic principles of vascular biology covered in an introductory physiology course. The design of this laboratory allows students to actively participate in an exercise demonstrating the regulation of arterial tone by endothelial and extrinsic factors. In addition, this hands-on laboratory allows students to gather data using well-known basic biomedical research techniques. Specifically, students are introduced to an isolated organ-chamber technique that is widely used to study cellular mechanisms of many tissues including vascular smooth muscle contraction and dilation. On the basis of student evaluations, participation in the experiments and interpreting data reinforce lecture materials on smooth muscle and endothelial cell function and illustrate mechanisms regulating vascular tone. Students come away with a greater understanding of vascular biology, a deeper appreciation of integrative physiology, and an understanding of the process of conducting tissue-chamber experiments.     

信息时代生态学本科专业微生物学实验课教学浅析

结合现代生态学本科专业的特点和学科发展需求,对微生物学实验课教学内容设置、实验课教学方法与效果以及课程考核办法进行了思考和探讨。科学地设置实验内容,优化利用学校多媒体网络技术,积极有效地安排实验课时间,充分调动学生的实验课兴趣,使学生更好地学习和掌握微生物学实验技能,为新世纪生态科学的发展培养优秀人才。     

Facile development of medium optimization for antibody production: implementation in spinner flask and hollow fiber reactor

Most bio-industrial mammalian cells are cultured in serum-free media to achieve advantages, such as batch consistency, suspended growth, and simplified purification. The successful development of a serum-free medium could contribute to a reduction in the experimental variation, enhance cell productivity, and facilitate biopharmaceuticals production using the cell culture process. Commercial serum-free media are also becoming more and more popular. However, the cell line secrets its own recombinant product and has special nutritional requirements. How can the composition of the proprietary medium be adjusted to support the specific cell’s metabolism and recombinant protein? This article uses statistical strategies to modify the commercial medium. A design of experiments is adopted to optimize the medium composition for the hybridoma cell in a serum-free condition. The supplements of peptone, ferric citrate, and trace elements were chosen to study their impact on hybridoma growth and antibody production using the response surface methodology. The stimulatory effect of the developed formulation on hybridoma growth was confirmed by the steepest ascent path. The optimal medium stimulated the hybridoma growth and antibody production in three diverse systems: a static plate, an agitated spinner flask, and a hollow fiber reactor. The cells in the developed serum-free medium had a better antibody production as compared to that in the commercial medium in the hollow fiber reactor. Our results demonstrated that the facile optimization for medium and antibody production was successfully accomplished in the hybridoma cells.     

本科植物细胞与基因工程研究型实验课程的构建与实践

在本科实验教学中开设研究型综合性实验课程是培养创新性人才的重要教改内容。作者利用自身的科研平台,为本科生开设了植物细胞与基因工程的专业选修课,主要讲授植物组织培养、原生质体分离与培养、植物遗传转化及转基因植物的筛选与鉴定等植物细胞与分子生物学技术原理及实验操作。通过整合植物组织、细胞和分子3个水平的实验教学内容和操作技术,采用集中讲解—独立操作—全天候开放的实验教学模式,并在实验教学中增加自主设计环节,培养本科生的综合性实验设计能力,训练独立操作技能,激发科研兴趣和自主创新意识,从而达到培养本科创新人才的目标。文章总结了开展植物细胞与基因工程实验教学的经验和教学效果,介绍了该实验教学的教学模式、教学内容和方法、考核评价体系,分析了存在的问题,阐述了以高水平科学研究促进实验教学对于深化本科教学改革和培养现代生物技术创新人才的重要性。     

Use of a Plackett-Burman statistical design to determine the effect of selected amino acids on monoclonal antibody production in CHO cells

Culture media design is central to the optimization of monoclonal antibody (mAb) production. Although general strategies do not currently exist for optimization of culture media, the combined use of statistical design and analysis of experiments and strategies based on simple material balances can facilitate culture media design. In this study, we evaluate the effect of selected amino acids on the growth rate and monoclonal antibody production of a Chinese hamster ovary DG-44 (CHO-DG44) cell line. These amino acids were selected based on their relative mass fraction in the specific mAb produced in this study, their consumption rate during bioreactor experiments, and also through a literature review. A Plackett-Burman statistical design was conducted to minimize the number of experiments needed to obtain statistically relevant information. The effect of this set of amino acids was evaluated during exponential cell culture (considering viable cell concentration and the specific growth rate as main output variables) and during the high cell-density stage (considering mAb final concentration and specific productivity as relevant output variables). For this particular cell line, leucine (Leu) and arginine (Arg) had the highest negative and positive effects on cell viability, respectively; Leu and threonine (Thr) had the highest negative effect on growth rate, and valine (Val) and Arg demonstrated the highest positive impact on mAb final concentration. Results suggest the pertinence of a two-stage strategy for amino acid supplementation, with a mixture optimized for cell growth and a different amino acid mixture for mAb production at high density.     

Bystander-mediated genomic instability after high LET radiation in murine primary haemopoietic stem cells

Communication between irradiated and unirradiated (bystander) cells can result in responses in unirradiated cells that are similar to responses in their irradiated counterparts. The purpose of the current experiment was to test the hypothesis that bystander responses will be similarly induced in primary murine stem cells under different cell culture conditions. The experimental systems used here, co-culture and media transfer, are similar in that they both restrict communication between irradiated and bystander cells to media borne factors, but are distinct in that with the media transfer technique, cells can only communicate after irradiation, and with co-culture, cells can communication before, during and after irradiation. In this set of parallel experiments, cell type, biological endpoint, and radiation quality and dose, were kept constant. In both experimental systems, clonogenic survival was significantly decreased in all groups, whether irradiated or bystander, suggesting a substantial contribution of bystander effects (BE) to cell killing. Genomic instability (GI) was induced under all radiation and bystander conditions in both experiments, including a situation where unirradiated cells were incubated with media that had been conditioned for 24h with irradiated cells. The appearance of delayed aberrations (genomic instability) 10-13 population doublings after irradiation was similar to the level of initial chromosomal damage, suggesting that the bystander factor is able to induce chromosomal alterations soon after irradiation. Whether these early alterations are related to those observed at later timepoints remains unknown. These results suggest that genomic instability may be significantly induced in a bystander cell population whether or not cells communicate during irradiation.     

创新性综合实验在食品科学专业人才培养中应用

以培养学生综合素质为指导思想,针对传统实验教学中的不足,在食品微生物实验中建立新的实验教学体系,验证性实验改为连续综合性实验,逐步引入创新设计性实验。微生物学实验改革使学生的综合能力得到了良好的训练,同时提供了实践的平台,学生可以将自己的创意通过创新性综合实验付诸实施,培养了创新意识和综合实验能力,收到良好的教学效果。