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In-line, Non-invasive, Non-destructive Cell Monitoring in Dynamically Growing Cultures



OBJECTIVE: To develop, design, and demonstrate a material product or device that will allow non-invasive, non-destructive, real-time, in-line monitoring of dynamically cultured cell viability and proliferation for GLP/GMP processes. 

DESCRIPTION: The Department of Defense has an urgent need for material products or devices that can operate in GLP/GMP processes that will allow for assessment of dynamically cultured cell viability and proliferation in ways that do not require invasive introduction of sampling equipment into the in-line production process or require extraction and destruction of samples. Cellular and tissue engineered therapies for regenerative medicine applications that do not derive from autologous sources must be processed in accordance with Good Manufacturing Practices prior to introduction into the body. This requires monitoring processes to assess various characteristics about the culture, e.g. cell viability, cell differentiation, and microbial contamination. In standard laboratory processes this would involve extraction of a sample for visual inspection, metabolic testing, or cell/tissue staining. From a GLP/GMP perspective, however, this is not an ideal methodology for several reasons. First, it introduces a foreign object into the production line (invasive), which increases the opportunity for contamination. Second, it terminally removes part of the product from the production line (destructive). Third, sample testing operates under the assumption that the region sampled is representative of the culture as a whole. However, while this assumption may be acceptable for 2D cultures, in 3D cultures, different regions may exhibit differential accumulations of metabolites or waste products. The currently available methods for monitoring the health of cultures in bioreactors typically involve indirect measurements of cell metabolism. Metabolic assays can evaluate cytotoxicity and drug effects [1], biocompatibility [2], viability [3], and proliferation. Some existing methodologies are already used for these assessments, and although they correlate reasonably well with many 2D cultures, they don’t correlate well with 3D cultures [4]. There is also some concern that they may be cytotoxic to some cell types [5]. Ultimately, direct measurements of cell health would be preferred. Another factor that influences the utility of a given measurement technique is whether or not it is suitable for cultures growing dynamically in a bioreactor. Standard cell culturing technique involves growing cells in flasks with a static overlaying medium containing the nutrients. This medium must be changed periodically as nutrients are used up and waste products accumulate. In a dynamically growing culture, media is flowed through a bioreactor, continually providing fresh nutrients and removing waste products. Although dynamically growing cultures do not necessarily preclude the use of a particular type of measurement, they will influence the applicability of a given method. The ultimate goal of this topic is to develop new material products that will allow direct assessment of dynamically growing cultured cell viability and proliferation without introducing an external sampling device (and the concomitant possibility of contamination) or the requisite destruction of the cells being measured.  

PHASE I: In the Phase I effort, innovative efforts for monitoring cell viability and proliferation will be conceptualized and designed. Such solutions may include visualization technologies, bioassays or other measuring devices. Solutions directed toward evaluation of 3D cultures are preferred, but exceptional studies that improve on the ability to non-invasively and non-destructively assess 2D cultures will be considered. Phase I efforts can support early concept work, or efforts necessary to support a regulatory submission. Proposed technologies or bioassays should be designed, and the development, fabrication or production procedures should be developed for a representative product. Cell types used should be obtained from a validated cell source such as Production Assistance for Cellular Therapies (PACT) at the National Heart, Lung, and Blood Institute. It is also recommended that in Phase 1 efforts offerors consider which measurement parameters are meaningful to industries, clinicians and regulatory bodies such as by conferring with the Standards Coordinating Body ( The Phase I effort should also include any fabrication experiments and benchmarking that demonstrate an adequate capability for meeting the expected challenges in fabricating or producing the proposed solution. Bioassays should be able to demonstrate lack of cytotoxicity and utility for the majority of cell types, particularly primary cells and adult stem cells. Specific milestones include the absence of cytotoxicity, the ability to show real-time changes to cell viability and proliferation, and any necessary algorithms for interpreting readouts. 

PHASE II: In the Phase II effort, a prototype technology should be fabricated and demonstrated. The performance of the technology or assay should be fully evaluated in terms of sensitivity, selectivity, dynamic range and limit of detection. Phase II results should demonstrate understanding of requirements to successfully enter Phase III, including how Phase II testing and validation will support independent user testing and qualification to lead towards use in a GLP/GMP environment.. Phase II studies may include work necessary to support a regulatory submission, to include, but not limited to: manufacturing development, qualification, packaging, stability, or sterility studies, etc. The researcher shall also describe in detail the transition plan for the Phase III effort.  

PHASE III: During phase III, it is envisioned that requirements to support a commercially available end item for use in a GMP manufacturing suite or for research will be completed. As part of that, scalability, repeatability and reliability of the proposed technology should be demonstrated. Devices should be fabricated using standard fabrication technologies and reliability. The proposal should include a commercialization or technology transition plan for the product that demonstrates how these requirements will be addressed including GMP manufacturing sufficient materials for evaluation. It is envisioned that this technology will be used in advanced regenerative medicine manufacturing efforts. Thus potential funding sources may be collaborative efforts with academia, other industries manufacturing consortia. This technology is envisioned for use in GLP/GMP facilities producing products for medical application. As such, the technology should have both military and civilian applications. Procurement of such technology would be at the discretion of the medical treatment facility. 


1: O'Brien J. Wilson I. Orton T. Pognan F. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem. 2000

2: 267:5421.

3:  Finch D.S. Oreskovic T. Ramadurai K. Herrmann C.F. George S.M. Mahajan R.L. Biocompatibility of atomic layer-deposited alumina thin films. J Biomed Mater Res Part A. 2008

4: 87A:100.

5:  Al-Nasiry S. Geusens N. Hanssens M. Luyten C. Pijnenborg R. The use of Alamar Blue assay for quantitative analysis of viability, migration and invasion of choriocarcinoma cells. Hum Reprod. 2007

6: 22:1304.

7:  Zhou X, Holsbeeks I, Impens S, Sonnaert M, Bloemen V, Luyten F, Schrooten J.Noninvasive real-time monitoring by alamarBlue(®) during in vitro culture of three-dimensional tissue-engineered bone constructs. Tissue Eng Part C Methods. 2013 Sep

8: 19(9):720-9.

9:  Mueller D. Tasher G. Damm G. Nüssler A.K. Heinzle E. Noor F. Real-time in situ viability assessment in a 3D bioreactor with liver cells using resazurin assay. Cytotechnology. 2013

10: 65:297.

KEYWORDS: Viability, Proliferation, In-line, Non-destructive, Non-invasive, Real-time 


Dr. Lloyd Rose 

(301) 619-8133 

Dr. Tony Gover 

(301) 619-9560 

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