Keywords: Cell Culture; Non-Invasive Optical Monitoring System

Novel Automated Nutrient Incorporation

Final Report

Abstract

The optimization of mammalian cell culture is vital for the growth of tissue that functions properly. We propose to develop a low-cost, on-line, non-invasive optical monitoring system to monitor the cell growth and viability during the cell culture. In this study, the device will include of light sources, light detectors and light guides. This will lead to the cell driven cell-feed bioprocess that can be continuously monitored without human intervention.

Keywords: Cell culture; non-invasive optical monitoring system.

Introduction

Many techniques used before to observe and measure the cell/tissue culture. However, monitoring for a small amount of sample has been a difficulty till now. The most commonly used approach is by using a glass electrode. This method requires a contact with a sample and it should be a relatively a large sample. It is thus not suitable for the measurement of the pH in small volumes as desired in cell culture [1]. So as to measure the sample with a small volume, other techniques have been developed. We also have the solid pH electrodes to measure the sample but it is not much reliable [2]. Another technique that is used widely is spectrophotometry that usually involves a dye that acts an indicator which is attached to the tip of an optical fiber. The disadvantage with this technique is that the pH probe has to be in contact with the sample solution directly which may lead to contamination of the probe tip leading to the decreased reliability of the system. Also with different media and different pH levels the dye acts differently and starts deteriorating with time. Finally, as the cells have to be monitored carefully, so the skilled person is required to be present which adds up to cost of the technique.

Theory

Generally the mammalian cell culture is feasible at pH ranging from 6.8 to 7.4. It is necessary to maintain the optimal conditions for the cell growth by maintaining the pH levels and replenishing the key nutrients periodically. The manual indulgence is required a lot of times so as to maintain the optimal conditions otherwise the automated cell monitoring/cultivation technique is used which is expensive. To overcome all above disadvantages we propose a low cost, non-invasive optical system that monitors the mammalian cell/tissue culture and requires less human intervention. In order to monitor the culture, our system will include the light sources, light detectors, optical fibers and the cell culture flasks. When using the spectral methods to determine the pH, artifacts such as concentration fluctuation of pH indicator can result in inaccuracy of measurement. To reduce the noise from the fluctuations in intensity of the excitation source we use the ratio metric techniques [3] instead of the simple intensity based measurements. Precisely the ratio of light intensities at two wavelengths (515 nm and 550 nm) will be measured in order to determine the pH value and to keep the costs low we will use the photo detectors, LED’s and the white light source in comparison to the UV-vis spectrometry. This technique allows us the on-line monitoring and the application of the feedback control systems to ensure the long term viability of the cell culture without further human intervention.

Fig. 1. Mammalian cells

Courtesy: Wikipedia

Experimental Setup

The setup will contain two photo detectors, two LEDs and optical fibers.

To reduce the noise we will instill ratio metric technique instead of the simple intensity based measurements. Specifically the ratio of two light intensities (515nm and 550nm) will be measured in order to determine the pH value. The method is cost effective as we use photo detectors and LED’s as compared to more expensive UV-spectrometry. Also non-invasive technique is more sterile and evolves no contamination of the fiber tip or the sample.

Fig.5. The experimental setup

Experimental Results

The media that will be used for the cell culture was tested for the different pH levels from 6 to 9. The pH was varied in the points of 0.5. The experiment was done using the UV-vis spectrometer (Perkin Elmer). The equipments and materials required were- a bottle of media (with a pH of 7.4), fluorescein-10µL, two cuvettes of 1cu cm volume. Additional components include pipette, beakers and kim wipes. The process starts by doing a background correction. A background correction was done by putting the undiluted medium of the same pH in both the cuvettes to set a reference zero (this should be done every time we change the pH). That means every time the pH of the solution is increased we set the reference. Take one of the cuvette out and then put 100µL fluorescein in it. Shake and mix it well so that the fluorescein does not stick on to the walls of the cuvette.

(a) (b)

Fig.2. (a) It contains the undiluted media, (b) 100µL fluorescein is added to the media

Fig. 3. The cuvettes are kept in the spectrometer for the scan.

Put it back in the UV-Vis Spectrometer to do a scan for absorbance by keeping the required parameters in check. Make sure that the wavelength range is between 350nm to 650 nm. Set the values for scanning like no. of nm/minute, wavelength and absorbance ranges. Repeat the same steps by varying the pH by 0.5. Save the data and plot it. The experimental duration is about 3 hours each time.

Fig. 4. The media with different pH has different colors.

From the experiment we found out that the absorbance curve for the different pH values (specifically 6 through 9) does not shift too much making the selection of the filter easier.

Fig. 4. The absorbance spectrum for the different pH values.

This process is done so as to facilitate the selection of appropriate filters for the setup.

It is easier to get the absorbance spectrum for the desired results as the absorbance is directly proportional to the path length, b, and the concentration, c, of the absorbing species. Beer's Law states that

A = bc,

Where  is a constant of proportionality, called the absorbtivity.

The point is that if we increase or decrease the concentration of the fluorescence than we can get the desired wavelengths patterns. Also we can calculate the same using the transmittance.

The same approach will be applied to the PEO (Polyethylene Oxide) hydrogels which have the ability to discharge key nutrients with the changing pH levels. They swell in water/ glucose. One such gel (that is to be test on) is shown below.

Fig. 5. PEO hydrogel in a dry state.

Future Approach

Future approach may contain of the lensed fiber as they don’t requite a fiber collimator and are more precise and cost effective.Lensed fibers [4] are high performance components for collimating and focusing light. Flexibility and precise control of lens geometry enable versatility in beam diameter and working distance. A monolithic device consisting of a Plano-convex refractive lens, fusion spliced to a single-mode optical fiber. This results in a collimator whose reliability and operating conditions are the same as those of the optical fiber.

Fig. 6. Some lensed fibers.

Courtesy: Corning

Fig. 7. The future setup

Courtesy: Dr. Wenhui Wang

Here the testing sample can be flown through a capillary tube and can have a reservoir elsewhere. This method will be best in terms of precision and cost effectiveness but right now as the cells and gels will more viscous so it can be thought as the later development stage of the project.

Summary

As the optical fibers will not be in contact with the sample so the contamination in this system can be avoided and thus providing the possibility of long-term on-line pH monitoring during cell/tissue culture. The setup for the NANI project is in finishing stages. Stabilization and calibration of the NANI gel is process.

Acknowledgement

This study was supported, mentored and assisted by Prof. Xingwei Wang of Department of Electrical Engineering, University of Massachusetts, Lowell.

Refererences

[1] Smith A, Pollard M, Cleaton-Jones P, Preston A. A comparison of

three electrodes for the measurement of pH in small volumes.

[2] Ahn BK, Liu CC, Neuman MR, Ko WK. Development of a miniature

pH electrode for biological applications.

[3] Putman RW. Intracellular pH regulation. In: Sperelakis N, editor.

Cell physiology source book.

[4] Dr. Wenhui Wang.

[5] An in line non-invasive optical system to monitor pH in cell and tissue culture

Xia Xua, Stanton Smith b, Jill Urban b, Zhanfeng Cui

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Introduction to biosensors