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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sterility+testing+for+hematopoietic+stem+cells.">Sterility testing for hematopoietic stem cells.</a> Journal of Clinical Microbiology. , (2023).</li><li>United States Food and Drug Administration. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidance+for+Industry.+Sterile+Drug+Products+Produced+by+Aseptic+Processing+-+Current+Good+Manufacturing+Practice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=USP&lt;71&gt;+Sterility+Tests+in+USP43-NF38.">USP&lt;71&gt; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+evaluation+of+compendial+USP&lt;71&gt;,+BacT/Alert+Dual-T,+and+Bactec+FX+for+detection+of+product+sterility+testing+contaminants.">Comprehensive evaluation of compendial USP&lt;71&gt;, 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sterility+testing+for+cellular+therapies:+What+is+the+role+of+the+clinical+microbiology+laboratory.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ISO+14644-1:2015+Cleanrooms+and+associated+controlled+environments+-+Part+1:+Classification+of+air+cleanliness+by+particle+concentration.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDA+Technical+Report+No.+13+Revised+2022+(TR+13)+Fundamentals+of+an+Environmental+Monitoring+Program.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=USP&lt;1116&gt;+Microbiological+Evaluation+of+Cleanrooms+and+Other+Controlled+Environments+in+USP43-NF38+2S.">USP&lt;1116&gt; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicenter+study+on+incubation+conditions+for+environmental+monitoring+and+aseptic+process+simulation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+different+incubation+conditions+for+microbiological+environmental+monitoring.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multisite+qualification+of+an+automated+incubator+and+colony+counter+for+environmental+and+bioburden+applications+in+pharmaceutical+microbiology.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=USP&lt;1072&gt;+Disinfectants+and+Antiseptics.">USP&lt;1072&gt; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=USP&lt;1071&gt;+Rapid+Microbial+Tests+for+Release+of+Sterile+Short-Life+Products:+A+Risk-Based+Approach+in+USP43-NF38+2S.">USP&lt;1071&gt; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PUSP&lt;1223&gt;+Validation+of+Alternative+Microbiological+Methods+in+USP43-NF38+2S.">PUSP&lt;1223&gt; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDA+Technical+Report+No.+33,+Revised+2013+(TR+33)+Evaluation,+Validation+and+Implementation+of+Alternative+and+Rapid+Microbiological+Methods.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+study+identifies+a+sensitive,+low-risk,+closed-system+model+for+detection+of+fungal+contaminants+in+cell+and+gene+therapy+products.">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 &amp; Pharmaceutical Inspection Co-operation. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guide+to+Good+Manufacturing+Practice+for+Medicinal+Products+Annexes+(PE++009-16+(Annexes)).">Guide to Good Manufacturing Practice for Medicinal Products Annexes (PE 009-16 (Annexes)).</a> Pharmaceutical Inspection Convention &amp; Pharmaceutical Inspection Co-operation Scheme. , (2022).</li><li>United States Pharmacopeia - National Formulary. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=USP&lt;825&gt;+Radiopharmaceuticals+-+Preparation,+Compounding,+Dispensing,+and+Repackaging+in+USP43-NF38+2S.">USP&lt;825&gt; 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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>20-25&#176;C Incubator</td>
			<td></td>
			<td></td>
			<td>Lab preference</td>
		</tr>
		<tr>
			<td>30-35&#176;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&#160;</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&#160;</td>
			<td>P595</td>
			<td>USP, irradiated</td>
		</tr>
		<tr>
			<td>Sabouraurd Dextrose Agar (SAB)</td>
			<td>Hardy Diagnostics&#160;</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&#160;</td>
			<td>W570R</td>
			<td>USP, irradiated</td>
		</tr>
		<tr>
			<td>Tryptic Soy Agar with Lecithinase and Tween (TSALT)</td>
			<td>Hardy Diagnostics&#160;</td>
			<td>P520R</td>
			<td>USP, irradiated</td>
		</tr>
		<tr>
			<td>Tryptic Soy Broth (TSB)</td>
			<td>Hardy Diagnostics&#160;</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&#160;</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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Research

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Bioengineering

3.2K 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  

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end organs. The latter include the lung, RES, and brain and are operative during human immunodeficiency virus type one (HIV-1) infection. Macrophage entry into tissues is notable in areas of active HIV-1 replication and sites of inflammation. In order to assess the potential of macrophages as nanocarriers, superparamagnetic iron-oxide and/or drug laden particles coated with surfactants were parenterally injected into HIV-1 encephalitic mice. This was done to quantitatively assess particle and drug biodistribution. Magnetic resonance imaging (MRI) test results were validated by histological coregistration and enhanced image processing. End organ disease as typified by altered brain histology were assessed by MRI. The demonstration of robust migration of nanoformulations into areas of focal encephalitis provides '"proof of concept" for the use of advanced bioimaging techniques to monitor macrophage migration. Importantly, histopathological aberrations in brain correlate with bioimaging parameters making the general utility of MRI in studies of cell distribution in disease feasible. We posit that using such methods can provide a real time index of disease burden and therapeutic efficacy with translational potential to humans.

Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end ...

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)9 addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.
A three-step pathway for alkane degradation was implemented in E. coli to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native &beta;-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2, rubA3, rubA4and rubB) of the alkane hydroxylase system from Gordonia sp. TF68,21 were transformed into E. coli. For the conversion of long-chain alkanes (C15-C36), theladA gene from Geobacillus thermodenitrificans was implemented. For the required further steps of the degradation process, ADH and ALDH (originating from G. thermodenitrificans) were introduced10,11. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.
To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.
The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFD&alpha; and &beta;, both from Pyrococcus horikoshii OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFD&alpha; and &beta; improve the protein folding process e.g. under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n-hexane in the culture medium were observed.
Summarizing, the results indicate that the toolkit enables E. coli to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.

A sustainable auto regulating bacterial system for the remediation of oil pollutions was designed using standard interchangeable DNA parts (BioBricks). An engineered E. coli strain was used to degrade alkanes via &beta;-oxidation in toxic aqueous environments. The respective enzymes from different species showed alkane degradation activity. Additionally, an increased tolerance to n-hexane was achieved by introducing genes from alkane-tolerant bacteria.

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular processes. There is a need for culturing and measuring systems that incorporate programmed illumination and stimulation of optogenetic systems. We present a protocol for building and using a continuous culturing apparatus to illuminate microbial cells with programmed doses of light, and automatically acquire and analyze images of cells in the effluent. The operation of this apparatus as a chemostat allows the growth rate and the cellular environment to be tightly controlled. The effluent of the continuous cell culture is regularly sampled and the cells are imaged by multi-channel microscopy. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses in the fluorescence intensity and cellular morphology of cells sampled from the culture effluent are measured over multiple days without user input. We demonstrate the utility of this culturing apparatus by dynamically inducing protein production in a strain of Saccharomyces cerevisiae engineered with an optogenetic system that activates transcription.

We designed a continuous culturing apparatus for use with optogenetic systems to illuminate cultures of microbes and regularly image cells in the effluent with an inverted microscope. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses to illumination can be measured over several days.

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular ...

Construction and Setup of a Bench-scale Algal Photosynthetic Bioreactor with Temperature, Light, and pH Monitoring for Kinetic Growth Tests

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic performance of microalgae-based biofuel production. Models that estimate microalgal growth under different conditions can help to optimize PBR design and operation. To be effective, the growth parameters used in these models must be accurately determined. Algal growth experiments are often constrained by the dynamic nature of the culture environment, and control systems are needed to accurately determine the kinetic parameters. The first step in setting up a controlled batch experiment is live data acquisition and monitoring. This protocol outlines a process for the assembly and operation of a bench-scale photosynthetic bioreactor that can be used to conduct microalgal growth experiments. This protocol describes how to size and assemble a flat-plate, bench-scale PBR from acrylic. It also details how to configure a PBR with continuous pH, light, and temperature monitoring using a data acquisition and control unit, analog sensors, and open-source data acquisition software.

This paper describes the assembly process and operation of a bench-scale photosynthetic bioreactor that can be used, in conjunction with other methods, to estimate pertinent kinetic growth parameters. This system continuously monitors the pH, light, and temperature using sensors, a data acquisition and control unit, and open-source data acquisition software.

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

Prospecting Microbial Strains for Bioremediation and Probiotics Development for Metaorganism Research and Preservation

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Globally, 4.5 billion people are economically dependent on the sea, where most of their livelihood is provided by coral reefs. Corals are of great importance and therefore their extinction leads to catastrophic consequences. There are several possible solutions to remediate marine pollutants and local contamination, including bioremediation. Bioremediation is the capacity of organisms to degrade contaminants. The approach presents several advantages, such as sustainability, relatively low cost, and the fact that it can be applied in different ecosystems, causing minimal impacts to the environment. As an extra advantage, the manipulation of endogenous microbiomes, including putative beneficial microorganisms for corals (pBMCs), may have probiotic effects for marine animals. In this context, the use of the two approaches, bioremediation and pBMC inoculation combined, could be promising. This strategy would promote the degradation of specific pollutants that can be harmful to corals and other metaorganisms while also increasing host resistance and resilience to deal with pollution and other threats. This method focuses on the selection of pBMCs to degrade two contaminants: the synthetic estrogen 17a-ethinylestradiol (EE2) and crude oil. Both have been reported to negatively impact marine animals, including corals, and humans. The protocol describes how to isolate and test bacteria capable of degrading the specific contaminants, followed by a description of how to detect some putative beneficial characteristics of these associated microbes to their coral host. The methodologies described here are relatively cheap, easy to perform, and highly adaptable. Almost any kind of soluble target compound can be used instead of EE2 and oil.

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Bioremediation is the capacity of organisms to degrade contaminants. Here, we describe methodologies to isolate and test microbes presenting bioremediation ability and potential probiotic characteristics for corals.

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. ...