This work was supported by the Intramural Research Program of the National Institutes of Health Clinical Center. The content is solely the responsibility of the authors and does not represent the official views of the National Institutes of Health.

Microbial Control and Monitoring Strategies for Cleanroom Environments

<ol><li>Cundell, T., Atkins, J. W., Lau, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sterility+testing+for+hematopoietic+stem+cells%5BTitle%5D)+AND+%22Journal+of+Clinical+Microbiology%22%5BJournal%5D)">Sterility testing for hematopoietic stem cells.</a> Journal of Clinical Microbiology. , 10.1128/jcm.01654-22 (2023).</li>
<li>United States Food and Drug Administration. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Guidance+for+Industry.+Sterile+Drug+Products+Produced+by+Aseptic+Processing+-+Current+Good+Manufacturing+Practice%5BTitle%5D)+AND+%22United+States+Food+and+Drug+Administration%22%5BJournal%5D)">Guidance for Industry. Sterile Drug Products Produced by Aseptic Processing - Current Good Manufacturing Practice.</a> United States Food and Drug Administration. , (2004).</li>
<li>United States Pharmacopeia - National Formulary. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((USP%3C71%3E+Sterility+Tests+in+USP43-NF38%5BTitle%5D)+AND+%22United+States+Pharmacopeia+-+National+Formulary%22%5BJournal%5D)">USP<71> Sterility Tests in USP43-NF38.</a> United States Pharmacopeia - National Formulary. , (2022).</li>
<li>England, M. R., Stock, F., Gebo, J. E. T., Frank, K. M., Lau, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comprehensive+evaluation+of+compendial+USP%3C71%3E%2C+BacT%2FAlert+Dual-T%2C+and+Bactec+FX+for+detection+of+product+sterility+testing+contaminants%5BTitle%5D)+AND+%22Journal+of+Clinical+Microbiology%22%5BJournal%5D)">Comprehensive evaluation of compendial USP<71>, BacT/Alert Dual-T, and Bactec FX for detection of product sterility testing contaminants.</a> Journal of Clinical Microbiology. 57 (2), 01548(2019).</li>
<li>Gebo, J. E. T., East, A. D., Lau, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+side-by-side+comparison+of+clinical+versus+current+good+manufacturing+practices+%28cGMP%29+microbiology+laboratory+requirements+for+sterility+testing+of+cellular+and+gene+therapy+products%5BTitle%5D)+AND+%22Clinical+Microbiology+Newsletter%22%5BJournal%5D)">A side-by-side comparison of clinical versus current good manufacturing practices (cGMP) microbiology laboratory requirements for sterility testing of cellular and gene therapy products.</a> Clinical Microbiology Newsletter. 43 (21), 181-191 (2021).</li>
<li>Gebo, J. E. T., Lau, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sterility+testing+for+cellular+therapies%3A+What+is+the+role+of+the+clinical+microbiology+laboratory%5BTitle%5D)+AND+%22Journal+of+Clinical+Microbiology%22%5BJournal%5D)">Sterility testing for cellular therapies: What is the role of the clinical microbiology laboratory.</a> Journal of Clinical Microbiology. 58 (7), 01492(2020).</li>
<li>International Organization for Standardization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ISO+14644-1%3A2015+Cleanrooms+and+associated+controlled+environments+-+Part+1%3A+Classification+of+air+cleanliness+by+particle+concentration%5BTitle%5D)+AND+%22International+Organization+for+Standardization%22%5BJournal%5D)">ISO 14644-1:2015 Cleanrooms and associated controlled environments - Part 1: Classification of air cleanliness by particle concentration.</a> International Organization for Standardization. , (2015).</li>
<li>Parenteral Drug Association. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PDA+Technical+Report+No.+13+Revised+2022+%28TR+13%29+Fundamentals+of+an+Environmental+Monitoring+Program%5BTitle%5D)+AND+%22Parenteral+Drug+Association%2C+Inc%22%5BJournal%5D)">PDA Technical Report No. 13 Revised 2022 (TR 13) Fundamentals of an Environmental Monitoring Program.</a> Parenteral Drug Association, Inc. , (2022).</li>
<li>United States Pharmacopeia - National Formulary. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((USP%3C1116%3E+Microbiological+Evaluation+of+Cleanrooms+and+Other+Controlled+Environments+in+USP43-NF38+2S%5BTitle%5D)+AND+%22United+States+Pharmacopeia+-+National+Formulary%22%5BJournal%5D)">USP<1116> Microbiological Evaluation of Cleanrooms and Other Controlled Environments in USP43-NF38 2S.</a> United States Pharmacopeia - National Formulary. , (2020).</li>
<li>Guinet, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multicenter+study+on+incubation+conditions+for+environmental+monitoring+and+aseptic+process+simulation%5BTitle%5D)+AND+%22PDA+journal+of+pharmaceutical+science+and+technology%22%5BJournal%5D)">Multicenter study on incubation conditions for environmental monitoring and aseptic process simulation.</a> PDA journal of pharmaceutical science and technology. 71 (1), 43-49 (2017).</li>
<li>Gordon, O., Berchtold, M., Staerk, A., Roesti, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+different+incubation+conditions+for+microbiological+environmental+monitoring%5BTitle%5D)+AND+%22PDA+Journal+of+Pharmaceutical+Science+and+Technology%22%5BJournal%5D)">Comparison of different incubation conditions for microbiological environmental monitoring.</a> PDA Journal of Pharmaceutical Science and Technology. 68 (5), 394-406 (2014).</li>
<li>Anders, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multisite+qualification+of+an+automated+incubator+and+colony+counter+for+environmental+and+bioburden+applications+in+pharmaceutical+microbiology%5BTitle%5D)+AND+%22PDA+Journal+of+Pharmaceutical+Science+and+Technology%22%5BJournal%5D)">Multisite qualification of an automated incubator and colony counter for environmental and bioburden applications in pharmaceutical microbiology.</a> PDA Journal of Pharmaceutical Science and Technology. , (2022).</li>
<li>United States Pharmacopeia - National Formulary. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((USP%3C1072%3E+Disinfectants+and+Antiseptics%5BTitle%5D)+AND+%22United+States+Pharmacopeia+-+National+Formulary%22%5BJournal%5D)">USP<1072> Disinfectants and Antiseptics.</a> United States Pharmacopeia - National Formulary. , (2020).</li>
<li>United States Pharmacopeia - National Formulary. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((USP%3C1071%3E+Rapid+Microbial+Tests+for+Release+of+Sterile+Short-Life+Products%3A+A+Risk-Based+Approach+in+USP43-NF38+2S%5BTitle%5D)+AND+%22United+States+Pharmacopeia+-+National+Formulary%22%5BJournal%5D)">USP<1071> Rapid Microbial Tests for Release of Sterile Short-Life Products: A Risk-Based Approach in USP43-NF38 2S.</a> United States Pharmacopeia - National Formulary. , (2020).</li>
<li>United States Pharmacopeia - National Formulary. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PUSP%3C1223%3E+Validation+of+Alternative+Microbiological+Methods+in+USP43-NF38+2S%5BTitle%5D)+AND+%22United+States+Pharmacopeia+-+National+Formulary%22%5BJournal%5D)">PUSP<1223> Validation of Alternative Microbiological Methods in USP43-NF38 2S.</a> United States Pharmacopeia - National Formulary. , (2020).</li>
<li>Parenteral Drug Association. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PDA+Technical+Report+No.+33%2C+Revised+2013+%28TR+33%29+Evaluation%2C+Validation+and+Implementation+of+Alternative+and+Rapid+Microbiological+Methods%5BTitle%5D)+AND+%22Parenteral+Drug+Association%2C+Inc%22%5BJournal%5D)">PDA Technical Report No. 33, Revised 2013 (TR 33) Evaluation, Validation and Implementation of Alternative and Rapid Microbiological Methods.</a> Parenteral Drug Association, Inc. , (2013).</li>
<li>Putnam, N. E., Lau, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comprehensive+study+identifies+a+sensitive%2C+low-risk%2C+closed-system+model+for+detection+of+fungal+contaminants+in+cell+and+gene+therapy+products%5BTitle%5D)+AND+%22Journal+of+Clinical+Microbiology%22%5BJournal%5D)">Comprehensive study identifies a sensitive, low-risk, closed-system model for detection of fungal contaminants in cell and gene therapy products.</a> Journal of Clinical Microbiology. 59 (11), 0135721(2021).</li>
<li>Pharmaceutical Inspection Convention & Pharmaceutical Inspection Co-operation. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Guide+to+Good+Manufacturing+Practice+for+Medicinal+Products+Annexes+%28PE++009-16+%28Annexes%29%29%5BTitle%5D)+AND+%22Pharmaceutical+Inspection+Convention+%26+Pharmaceutical+Inspection+Co-operation+Scheme%22%5BJournal%5D)">Guide to Good Manufacturing Practice for Medicinal Products Annexes (PE 009-16 (Annexes)).</a> Pharmaceutical Inspection Convention & Pharmaceutical Inspection Co-operation Scheme. , (2022).</li>
<li>United States Pharmacopeia - National Formulary. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((USP%3C825%3E+Radiopharmaceuticals+-+Preparation%2C+Compounding%2C+Dispensing%2C+and+Repackaging+in+USP43-NF38+2S%5BTitle%5D)+AND+%22United+States+Pharmacopeia+-+National+Formulary%22%5BJournal%5D)">USP<825> Radiopharmaceuticals - Preparation, Compounding, Dispensing, and Repackaging in USP43-NF38 2S.</a> United States Pharmacopeia - National Formulary. , (2020).</li>
</ol>

The authors declare no conflicts of interest.

There are several critical areas in this protocol, including the maintenance of aseptic technique and unidirectional airflow within cleanrooms and the BSCs. Best practices include moving slowly and deliberately to minimize turbulence. Aseptic manipulations should be performed from the side of the product, not from above. Closed system processing and the use of terminally sterilized raw materials are recommended. Speaking in critical areas and leaning against walls or equipment should be avoided. Similarly, unnecessary to...

Implementing a strong microbial monitoring program through environmental monitoring (EM), process monitoring, and product sterility testing is a regulatory requirement for current good tissue practices (cGTP) and current good manufacturing practices (cGMP) in cellular therapy laboratories1. Additionally, the United States Food and Drug Administration (FDA) expects that the laboratory performing the quality control (QC) testing of the product should also employ facilities and controls comparable to those used for aseptic filling operations2. 
This protocol has four main sections: 1) Aseptic practices, including personnel gowning, cleaning, and staging of materials; 2) EM, including viable air and surface cultures and non-viable particle air monitoring; 3) process monitoring, including settling plates and gloved fingertip sampling; and 4) product sterility testing via the compendial United States Pharmacopeia (USP) &lt;71&gt; method3 or the NIH Alternative Sterility Testing Method4. When used together, these measures can be an effective method for ensuring that a facility remains in a state of control.
The techniques described here are not novel; however, current standards from regulators and professional organizations lack detail, which has led to an absence of microbial monitoring or the implementation of non-standardized practices, particularly in academic centers where on-site manufacturing and product sterility testing are emerging at a rapid rate1,5,6. This protocol can be used as a guide to create a microbial monitoring and control program that meets regulatory requirements when used in conjunction with end-user validation and risk assessments.

<table><tbody><tr><td>20-25&amp;deg;C Incubator</td><td></td><td></td><td>Lab preference</td></tr><tr><td>30-35&amp;deg;C Incubator</td><td></td><td></td><td>Lab preference</td></tr><tr><td>Alcohol-based hand sanitizer</td><td></td><td></td><td>Lab preference</td></tr><tr><td>BacT/ALERT Dual-T instrument</td><td>BioMerieux Industry</td><td></td><td></td></tr><tr><td>Beard cover</td><td></td><td></td><td>Lab preference</td></tr><tr><td>Biosafety cabinet (BSC)</td><td></td><td></td><td>Lab preference</td></tr><tr><td>Cleanroom shoes</td><td></td><td></td><td>Lab preference</td></tr><tr><td>Fluidthioglycollate medium (FTM)</td><td>Hardy Diagnostics&amp;nbsp;</td><td>U84</td><td>USP</td></tr><tr><td>Handheld cleaning mop</td><td>Contec</td><td>2665LF</td><td></td></tr><tr><td>Hypodermic needle</td><td></td><td></td><td>Lab preference</td></tr><tr><td>iFA+ BacT/ALERT bottle</td><td>Biomerieux</td><td>412990</td><td></td></tr><tr><td>iFN+ BacT/ALERT bottle</td><td>Biomerieux</td><td>412991</td><td></td></tr><tr><td>Isokinetic head</td><td></td><td></td><td>Lab preference</td></tr><tr><td>Laser particle counter</td><td>TSI Incorporated</td><td>9500-01</td><td></td></tr><tr><td>LpH III</td><td>Steris</td><td>1S16CX</td><td></td></tr><tr><td>Mirror</td><td></td><td></td><td>Lab preference</td></tr><tr><td>Non-sterile bouffant</td><td></td><td></td><td>Lab preference</td></tr><tr><td>Non-sterile gloves</td><td></td><td></td><td>Lab preference</td></tr><tr><td>Non-sterile shoe covers</td><td></td><td></td><td>Lab preference</td></tr><tr><td>Non-sterile sleeve covers</td><td></td><td></td><td>Lab preference</td></tr><tr><td>Parafilm</td><td></td><td></td><td>Lab preference</td></tr><tr><td>Peridox RTU</td><td>Contec</td><td>CR85335IR</td><td></td></tr><tr><td>Plastic bag</td><td></td><td></td><td>Lab preference</td></tr><tr><td>Sabouraud Dextrose Agar with Lecithinase and Tween (SABLT)</td><td>Hardy Diagnostics&amp;nbsp;</td><td>P595</td><td>USP, irradiated</td></tr><tr><td>Sabouraurd Dextrose Agar (SAB)</td><td>Hardy Diagnostics&amp;nbsp;</td><td>W565</td><td>USP, irradiated</td></tr><tr><td>Safety glasses</td><td></td><td></td><td>Lab preference</td></tr><tr><td>Scrubs (top and bottom)</td><td></td><td></td><td>Lab preference</td></tr><tr><td>Spor-Klenx RTU</td><td>Steris</td><td>6525M2</td><td></td></tr><tr><td>Sterile 70% isopropyl alcohol (IPA)</td><td>Decon CiDehol</td><td>8316</td><td></td></tr><tr><td>Sterile alcohol wipe</td><td></td><td></td><td>Lab preference</td></tr><tr><td>Sterile boot covers</td><td>Kimberly Clark</td><td></td><td>Cat# varies based on size</td></tr><tr><td>Sterile coveralls</td><td>Kimberly Clark</td><td></td><td>Cat# varies based on size</td></tr><tr><td>Sterile face mask</td><td></td><td></td><td>Lab preference</td></tr><tr><td>Sterile gloves</td><td></td><td></td><td>Lab preference</td></tr><tr><td>Sterile hood</td><td>Kimberly Clark</td><td></td><td>Cat# varies based on size</td></tr><tr><td>Sterile low-lint wipes</td><td>Texwipe</td><td>TX3210</td><td></td></tr><tr><td>Sterile mop cleaning pads</td><td>Contec</td><td>MEQT0002SZ</td><td></td></tr><tr><td>Sterile sleeve covers</td><td>Kimberly Clark</td><td>36077</td><td></td></tr><tr><td>Sterile spreading rod</td><td>Fisher Scientific</td><td>14665231</td><td></td></tr><tr><td>Sterile syringe</td><td></td><td></td><td>Lab preference</td></tr><tr><td>Tacky mats</td><td></td><td></td><td>Lab preference</td></tr><tr><td>Tryptic Soy Agar (TSA)</td><td>Hardy Diagnostics&amp;nbsp;</td><td>W570R</td><td>USP, irradiated</td></tr><tr><td>Tryptic Soy Agar with Lecithinase and Tween (TSALT)</td><td>Hardy Diagnostics&amp;nbsp;</td><td>P520R</td><td>USP, irradiated</td></tr><tr><td>Tryptic Soy Broth (TSB)</td><td>Hardy Diagnostics&amp;nbsp;</td><td>U46</td><td>USP</td></tr><tr><td>Tubing</td><td></td><td></td><td>Lab preference</td></tr><tr><td>Vesphene III</td><td>Steris</td><td>1S15CX</td><td></td></tr><tr><td>Viable air sampler</td><td>Hardy Diagnostics&amp;nbsp;</td><td>BAS22K</td><td></td></tr><tr><td>Vortex</td><td></td><td></td><td>Lab preference</td></tr></tbody></table>

microbial control and monitoring strategies for cleanroom environments and cellular therapies

A well-validated and holistic program that incorporates robust gowning, cleaning, environmental monitoring, and personnel monitoring measures is critical for minimizing the microbial bioburden in cellular therapy manufacturing suites and the corresponding testing laboratories to ensure that the facilities are operating in a state of control. Ensuring product safety via quality control measures, such as sterility testing, is a regulatory requirement for both minimally manipulated (section 361) and more than minimally manipulated (section 351) human cells, tissues, and cellular and tissue-based products (HCT/Ps). In this video, we provide a stepwise guide for how to develop and incorporate the best aseptic practices for operating in a cleanroom environment, including gowning, cleaning, staging of materials, environmental monitoring, process monitoring, and product sterility testing using direct inoculation, provided by the United States Pharmacopeia (USP&lt;71&gt;) and the National Institutes of Health (NIH) Alternative Sterility Testing Method. This protocol is intended as a reference guide for establishments expected to meet current good tissue practices (cGTP) and current good manufacturing practices (cGMP).

The expected results are described in Table 1. The EM data should be reviewed and followed up with an appropriate investigation and response to action, alert, or ISO limit excursions. If an excursion occurs for non-viable particles, one should proceed as per ISO 14644-Annex A, sec A.5.57. If the excursion can be attributed to an immediately identifiable abnormal occurrence, the original sampling results should be documented, a note should be added to disregard the original results...

Watch this Scientific Journal Video about Microbial Control and Monitoring Strategies for Cleanroom Environments and Cellular Therapies at JoVE.com

Author Spotlight: Microbial Control and Monitoring Strategies for Cleanroom Environments and Cellular Therapies  

The protocol summarizes the best practices to minimize microbial bioburden in a cleanroom environment and includes strategies such as environmental monitoring, process monitoring, and product sterility testing. It is relevant for manufacturing and testing facilities that are required to meet current good tissue practice standards and current good manufacturing practice standards.

Microbial Control and Monitoring Strategies for Cleanroom Environments and Cellular Therapies

A well-validated and holistic program that incorporates robust gowning, cleaning, environmental monitoring, and personnel monitoring measures is critical ...

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3.3K Views.  National Institutes of Health. The protocol summarizes the best practices to minimize microbial bioburden in a cleanroom environment and includes strategies such as environmental monitoring, process monitoring, and product sterility testing. It is relevant for manufacturing and testing facilities that are required to meet current good tissue practice standards and current good manufacturing practice standards. 

Video: Author Spotlight: Microbial Control and Monitoring Strategies for Cleanroom Environments and Cellular Therapies  

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

Developmental Biology

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized medicine innovations. However, translating these advances into therapeutic applications will require in vitro systems that allow for robust, flexible, and cost effective bioreactor systems. There are several bioreactor systems currently utilized in research and commercial settings; however, many of these systems are not optimal for establishing, expanding, and monitoring the growth of different cell types. The culture parameters most challenging to control in these systems include, minimizing hydrodynamic shear, preventing nutrient gradient formation, establishing uniform culture medium aeration, preventing microbial contamination, and monitoring and adjusting culture conditions in real-time. Using a pneumatic single-use bioreactor system, we demonstrate the assembly and operation of this novel bioreactor for mammalian cells grown on micro-carriers. This bioreactor system eliminates many of the challenges associated with currently available systems by minimizing hydrodynamic shear and nutrient gradient formation, and allowing for uniform culture medium aeration. Moreover, the bioreactor&#8217;s software allows for remote real-time monitoring and adjusting of the bioreactor run parameters. This bioreactor system also has tremendous potential for scale-up of adherent and suspension mammalian cells for production of a variety therapeutic proteins, monoclonal antibodies, stem cells, biosimilars, and vaccines.

Using a pneumatic bioreactor, we demonstrate the assembly, operation, and performance of this single-use bioreactor system for the growth of mammalian cells.

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Automated Counterflow Centrifugal System for Small-Scale Cell Processing

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and product concentration are essential components for most manufacturing processes. However, at the early stages of product development, these steps are often performed manually. Manual dead-end centrifugation for buffer exchange is labor-intensive, costly, and not scalable. A closed automated system can effectively eliminate this laborious step, but implementation can be challenging. Here, we describe a newly developed cell processing device that is suitable for small- to medium-scale cell processing and aims to bridge the gap between manual processing and large-scale automation. This protocol can be easily applied to various cell types and processes by modifying the flow rate and centrifugation speed. Our protocol demonstrated high cell recovery with shorter processing times in comparison to the manual process. Cells recovered from the automated process also maintained their proliferation rates. The device can be applied as a modular component in a closed manufacturing process to accommodate steps such as buffer exchange, cell formulation, and cryopreservation.

Automation is key to upscaling and cost management in cell manufacturing. This manuscript describes the use of a counterflow centrifugal cell processing device for automating the buffer exchange and cell concentration steps for small-scale bioprocessing.

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and ...

Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to develop practical technologies that couple microscopic cell screening with cell isolation at high purity for downstream genetic analysis. Here, the use of photodegradable poly(ethylene glycol) hydrogels for screening and isolation of bacteria with unique growth phenotypes from heterogeneous cell populations is described. The method relies on encapsulating or entrapping cells with the hydrogel, followed by culture, microscopic screening, then use of a high-resolution light patterning tool for spatiotemporal control of hydrogel degradation and release of selected cells into a solution for retrieval. Applying different light patterns allows for control over the morphology of the extracted cell, and patterns such as rings or crosses can be used to retrieve cells with minimal direct UV light exposure to mitigate DNA damage to the isolates. Moreover, the light patterning tool delivers an adjustable light dose to achieve various degradation and cell release rates. It allows for degradation at high resolution, enabling cell retrieval with micron-scale spatial precision. Here, the use of this material to screen and retrieve bacteria from both bulk hydrogels and microfabricated lab-on-a-chip devices is demonstrated. The method is inexpensive, simple, and can be used for common and emerging applications in microbiology, including isolation of bacterial strains with rare growth profiles from mutant libraries and isolation of bacterial consortia with emergent phenotypes for genomic characterizations.

The use of photodegradable hydrogels to isolate bacterial cells by utilizing a high-resolution light pattering tool is reported. Essential experimental procedures, results, and advantages of the process are reviewed. The method enables rapid and inexpensive isolation of targeted bacteria showing rare or unique functions from heterogeneous communities or populations.

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to ...

Automated and High-throughput Microbial Monoclonal Cultivation and Picking Using the Single-cell Microliter-droplet Culture Omics System

Pure bacterial cultures are essential for the study of microbial culturomics. Traditional methods based on solid plates, well plates, and micro-reactors are hindered by cumbersome procedures and low throughput, impeding the rapid progress of microbial culturomics research. To address these challenges, we had successfully developed the Single-cell Microliter-droplet Culture Omics System (MISS cell), an automated high-throughput platform that utilizes droplet microfluidic technology for microbial monoclonal isolation, cultivation, and screening. This system can generate a large number of single-cell droplets and cultivate, screen, and collect monoclonal colonies in a short time, facilitating an integrated process from microbial isolation to picking. In this protocol, we demonstrated its application using the isolation and cultivation of human gut microbiota as an example and compared the microbial isolation efficiency, monoclonal culture performance, and screening throughput using the solid-plate culture method. The experimental workflow was simple, and reagent consumption was very low. Compared to solid-plate culture methods, the MISS cell could cultivate a greater diversity of gut microbiota species, offering significant potential and value for microbial culturomics research.

This protocol describes how to use the Single-cell Microliter-droplet Culture Omics System (MISS cell) to perform microbial monoclonal isolation, cultivation, and picking. The MISS cell achieves an integrated workflow based on droplet microfluidic technology, which offers excellent droplet monodispersity, high parallel cultivation, and high-throughput biomass detection.

Pure bacterial cultures are essential for the study of microbial culturomics. Traditional methods based on solid plates, well plates, and micro-reactors ...

Operational Protocols for Handling Wildling Mice in Research Facilities

Operating and Biocontainment Procedures of a Facility for Laboratory Mice with a Natural Microbiome: Immunophenotyping Procedure

The use of laboratory mice with a natural microbiome, such as &#34;Wildling mice&#34;, offers a promising research tool for both basic and applied science due to their close resemblance to the human superorganism. However, the breeding and maintenance of these mice, which harbor a diverse microbiome&#160;including bacteria, viruses, and parasites, pose significant challenges for animal husbandry facilities at research institutions. To address these challenges, a specialized facility concept was developed for housing &#34;Wildling mice&#34; at Charit&#233; - Universit&#228;tsmedizin Berlin. This approach involved designing a facility with specific structural features and operational protocols to effectively contain the natural microbiome, thereby protecting areas with higher hygiene standards.
A methodology for blood sampling from both specified pathogen-free (SPF) and &#34;Wildling mice&#34; for immunophenotyping is demonstrated, highlighting the workflow and biocontainment measures implemented in the facility. Remarkable results reveal that &#34;Wildling mice&#34; exposed to a natural microbiome develop distinct immune cell populations, which are significantly reduced in mice bred and maintained under stringent hygiene conditions.
The significance of this study lies in its potential to provide researchers with access to mice that possess a natural microbiome and a mature immune system similar to that of human adults. This approach could enhance the translatability of preclinical findings into clinical practice, thereby advancing the field of biomedical research.

Here, we describe the structure and operating procedures, including microbial containment measures of a facility for "Wilding mice" using blood sampling for immunophenotyping as an example.

The use of laboratory mice with a natural microbiome, such as &#34;Wildling mice&#34;, offers a promising research tool for both basic and applied science ...

Immunology and Infection

Cell Culture Techniques and Practices to Avoid Contamination by Fungi and Bacteria in the Research Cell Culture Laboratory

Cell culture is a delicate skill necessary for growing human, animal, and insect cells, or other tissues, in a controlled environment. The goal of the protocol is to emphasize the correct techniques used in a research laboratory to prevent contamination from fungi and bacteria. Special emphasis is placed on avoiding mycoplasma contamination, a major concern in the cell culture room due to its small size and resistance to most antibiotics used for cell culture. These same techniques ensure continuous growth and maintain healthy cells. For new and experienced cell culture users alike, it&#8217;s important to consistently adhere to these best practices to mitigate the risk of contamination. Once a year, laboratories should review cell culture best practices and follow-up with a discussion or additional training if needed. Taking early action to prevent contamination in the first place will save time and money, as compared to cleaning up after contamination occurs. Universal best practices keep cell cultures healthy, thereby reducing the need to constantly thaw new cells, purchase expensive cell culture media, and reducing the amount of incubator decontamination and downtime.&#160;

This protocol presents essential cell culture techniques and practices to be used in the research cell culture laboratory to avoid contamination by fungi and bacteria. Within the category of bacteria, special emphasis will be placed on preventing mycoplasma contamination.&#160;&#160;

Cell culture is a delicate skill necessary for growing human, animal, and insect cells, or other tissues, in a controlled environment. The goal of the ...