**1. Introduction**

#### **1.1. Motivation for cell sorting**

The cell is the basic functional unit within a tissue or an organ. Methods that can be used to probe the cell, so as to understand, or even manipulate its interrelated processes, pathways, and/or overall functioning, are of great scientific and commercial value. Research efforts in molecular biology, biochemistry, and biotechnology over the last two decades have created high demand for efficient, cost-effective, cell enrichment, isolation, and handling methods. Cell studies can be performed on continuously growing cell lines, many of which are commercially available, in tissue culture, or on cells obtained from intact tissues or isolated from blood [1-3].

Mammalian cells are highly heterogeneous in structure, function, and characteristics. However, many types of biochemical, pharmaceutical, and clinical studies, such as immunophenotyping, studies of the cell cycle, cell proliferation, or apoptosis, and other specialized cell analyses require a homogenous population consisting of a single cell type; as the analyte. Only then can the results deemed accurate and specific to the cell type under investigation [4, 5]. Accordingly, techniques to separate cell types in a heterogeneous cell population are of immense practical value. Any such efforts are further complicated when the target cell is rare within a population such as in many cancer and prenatal diagnosis applications. The more stringent the requirements for specific and precise cell separation, the greater the degree of accuracy and reproducibility required in the technology that underlies the separation method [6-8].

Recent progress in microfabrication, technologies developed and utilized by the integrated circuits (ICs) industry, is being exploited to biomedicine, spawning a relatively new field of research that has become known as BioMEMS. Microfabricated devices have already had a

© 2012 Mohamed, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Mohamed, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

broad range of biomedical and biological applications [9, 10]. These devices can be manufactured with a reproducible accuracy of less than 1 micrometer (1/100th the diameter of a human hair). In the last decade, microchips have been used in a huge range of devices and contexts: microscale sensors for surgical instruments, monitoring of physiological activities, drug discovery and delivery, DNA amplification, and electrophoresis, as well as cell sorting, the application discussed in this chapter [11-16].

Use of Microfluidic Technology for Cell Separation 197

denotes a

shape. The change in shape and the increased rigidity of the RBCs lead to obstruction of capillaries, thereby restricting blood flow to organs, causing anemia and other "sickle cell crisis", and decreasing life expectancy. Cancer is a second example of such changes [20]. Malignant tumor cells differ from benign tumor cells in structure, growth rate, invasiveness, and their ability to metastasize. Benign tumor cells grow slowly, pushing neighboring tissues away while staying well encapsulated. In contrast, malignant tumor cells grow rapidly, invade neighboring tissues, and may metastasize [21]. Furthermore, they generally are irregular in shape, and exhibit a more "rugged" or "ruffled" surface appearance than do normal cells [22, 23]. Cancer is detected and diagnosed based on physical changes present in tissues and cells. Nuclear changes such as an increase in size, deformation, and a change in internal organization are among the most universal criteria for detecting malignancy [24-25]. These changes may reflect alterations in the nuclear matrix and the connections of the nuclear matrix to or via the cytoskeleton. Cancer cells modify their morphology, principally by increasing the size of the nucleus, before they become invasive, i.e., in dysplasia and carcinoma *in situ*. The nucleus of a dysplastic cell can be up to four times as large as that of a non-dysplastic cell. Accordingly, light scattering microscopy has been used to distinguish normal, dysplastic, and cancerous epithelial cells in a range of tissues including esophagus, colon, bladder, and oral cavity [26]. However, this technique can only succeed if a sufficient

Additionally, most cell types have characteristic complements of surface molecules. Among the most useful for identification is the cluster of differentiation (CD), or cluster of designation. CD molecules have roles in signaling and adhesion processes, and the specific compliments of CD molecules is a determinant of the specific function of the cell. Cell populations are usually denoted by a pattern of "+" or a "-"scorings; indicating the presence

cell that expresses CD34 but not CD31. Use of more than one marker can make the cell selection very specific. However, not all cell types have a known specific surface marker

For some cells such as cancer cells, not many cell-surface markers have been identified. In such a situation, some techniques have used a surface marker directed against surface membrane antigens that are expressed in tissue of origin, which is most often epithelial. Thus, detection of these tissue-specific surface markers in the blood stream is suggesting that cancer cells have detached from the tumor. Some techniques attempting to isolate circulating tumor cells (CTCs) from whole blood have used EpCAM, an epithelial cell adhesion molecule that is overexpressed in epithelial carcinomas such as colon and breast. However, published studies have shown inconsistent frequency of EpCAM expression in breast cancer, from as low as 35% to as high as 100% [28-31]. Thus, EpCAM cannot be considered a CTC specific marker. Additional bio-molecules such as DAPI (nucleic acid stain) and antibodies against cytokeratin (expressed on the epithelial cell membrane) and CD45 (expressed on the majority of hematopoietic cells) must be used if captured cells are to be positively identified as CTC. Therefore a cell with the phenotype EpCAM+, DAPI+, CK+,

or absence of specific CD molecules. For example, a nomenclature of CD34+, CD31-

number of cells are available for analysis.

and CD45- is considered a CTC.

[27].

Microfluidic technology, a subcategory of BioMEMS, is a set of techniques and processes for making devices to precisely control and manipulate fluid in a geometrically small channels; sub- to few hundred- micrometers in size. Microfluidic is multi-disciplinary; developing a device with biological utility requires the integration of knowledge and techniques from the fields of engineering, biology, physics, and chemistry. Such microfabricated devices are used to study biological systems and to generate new insights into how these systems work. Conversely, the biological knowledge gained through micro/nano - scale analyses can lead to further improvements in device design. BioMEMS is a challenging field because the materials, and chemistries, important for biological microfluidics applications are so diverse [17, 18].

The objective of the present chapter is to introduce the principals of cell sorting by microfluidic technology, and to discuss its strengths, current limitations, and current and potential applications, with illustrative examples from the literature and from the author's laboratory.

#### **1.2. Challenges in cell sorting**

Cells of different types have characteristic sizes, shapes, densities, and arrays of surface molecules that can be exploited for sorting. For example, red blood cells (RBCs) are the cells responsible for delivering oxygen to the tissues. RBCs have to squeeze through capillaries and therefore are relatively small, approximately 6-8 µm in diameter, and flexible. Mature RBCs are anucleate; the maturing cells lose their nuclei before leaving the bone marrow. Loss of the nucleus leads to cell membrane collapse, conferring the characteristic biconcave shape of RBCs, and giving the cell a greater surface area to volume ratio than of spherical cells. These physical features allow easier movement of oxygen (O2) and carbon dioxide (CO2) through the membrane. RBCs are composed mainly of hemoglobin, whose iron atoms temporarily bind O2 molecules in the lungs, and then release the molecules throughout the body. RBCs have the highest density of any cell type in blood. All of the above characteristics can be used for separation: for example size can be used to separate RBCs since they are smaller and more flexible than white blood cells (WBCs). RBCs' high density causes them to spin down to the bottom of a test tube after density gradient centrifugation. The high iron content gives RBCs intrinsic magnetic properties and can be used for magnetic separation [19].

In other cases, a cell changes its shape or size as a result to a disease or change in function. In sickle-cell disease, a genetic blood disorder, RBCs assume an abnormal, rigid, sickle shape. The change in shape and the increased rigidity of the RBCs lead to obstruction of capillaries, thereby restricting blood flow to organs, causing anemia and other "sickle cell crisis", and decreasing life expectancy. Cancer is a second example of such changes [20]. Malignant tumor cells differ from benign tumor cells in structure, growth rate, invasiveness, and their ability to metastasize. Benign tumor cells grow slowly, pushing neighboring tissues away while staying well encapsulated. In contrast, malignant tumor cells grow rapidly, invade neighboring tissues, and may metastasize [21]. Furthermore, they generally are irregular in shape, and exhibit a more "rugged" or "ruffled" surface appearance than do normal cells [22, 23]. Cancer is detected and diagnosed based on physical changes present in tissues and cells. Nuclear changes such as an increase in size, deformation, and a change in internal organization are among the most universal criteria for detecting malignancy [24-25]. These changes may reflect alterations in the nuclear matrix and the connections of the nuclear matrix to or via the cytoskeleton. Cancer cells modify their morphology, principally by increasing the size of the nucleus, before they become invasive, i.e., in dysplasia and carcinoma *in situ*. The nucleus of a dysplastic cell can be up to four times as large as that of a non-dysplastic cell. Accordingly, light scattering microscopy has been used to distinguish normal, dysplastic, and cancerous epithelial cells in a range of tissues including esophagus, colon, bladder, and oral cavity [26]. However, this technique can only succeed if a sufficient number of cells are available for analysis.

196 Blood Cell – An Overview of Studies in Hematology

[17, 18].

laboratory.

separation [19].

**1.2. Challenges in cell sorting** 

cell sorting, the application discussed in this chapter [11-16].

broad range of biomedical and biological applications [9, 10]. These devices can be manufactured with a reproducible accuracy of less than 1 micrometer (1/100th the diameter of a human hair). In the last decade, microchips have been used in a huge range of devices and contexts: microscale sensors for surgical instruments, monitoring of physiological activities, drug discovery and delivery, DNA amplification, and electrophoresis, as well as

Microfluidic technology, a subcategory of BioMEMS, is a set of techniques and processes for making devices to precisely control and manipulate fluid in a geometrically small channels; sub- to few hundred- micrometers in size. Microfluidic is multi-disciplinary; developing a device with biological utility requires the integration of knowledge and techniques from the fields of engineering, biology, physics, and chemistry. Such microfabricated devices are used to study biological systems and to generate new insights into how these systems work. Conversely, the biological knowledge gained through micro/nano - scale analyses can lead to further improvements in device design. BioMEMS is a challenging field because the materials, and chemistries, important for biological microfluidics applications are so diverse

The objective of the present chapter is to introduce the principals of cell sorting by microfluidic technology, and to discuss its strengths, current limitations, and current and potential applications, with illustrative examples from the literature and from the author's

Cells of different types have characteristic sizes, shapes, densities, and arrays of surface molecules that can be exploited for sorting. For example, red blood cells (RBCs) are the cells responsible for delivering oxygen to the tissues. RBCs have to squeeze through capillaries and therefore are relatively small, approximately 6-8 µm in diameter, and flexible. Mature RBCs are anucleate; the maturing cells lose their nuclei before leaving the bone marrow. Loss of the nucleus leads to cell membrane collapse, conferring the characteristic biconcave shape of RBCs, and giving the cell a greater surface area to volume ratio than of spherical cells. These physical features allow easier movement of oxygen (O2) and carbon dioxide (CO2) through the membrane. RBCs are composed mainly of hemoglobin, whose iron atoms temporarily bind O2 molecules in the lungs, and then release the molecules throughout the body. RBCs have the highest density of any cell type in blood. All of the above characteristics can be used for separation: for example size can be used to separate RBCs since they are smaller and more flexible than white blood cells (WBCs). RBCs' high density causes them to spin down to the bottom of a test tube after density gradient centrifugation. The high iron content gives RBCs intrinsic magnetic properties and can be used for magnetic

In other cases, a cell changes its shape or size as a result to a disease or change in function. In sickle-cell disease, a genetic blood disorder, RBCs assume an abnormal, rigid, sickle Additionally, most cell types have characteristic complements of surface molecules. Among the most useful for identification is the cluster of differentiation (CD), or cluster of designation. CD molecules have roles in signaling and adhesion processes, and the specific compliments of CD molecules is a determinant of the specific function of the cell. Cell populations are usually denoted by a pattern of "+" or a "-"scorings; indicating the presence or absence of specific CD molecules. For example, a nomenclature of CD34+, CD31 denotes a cell that expresses CD34 but not CD31. Use of more than one marker can make the cell selection very specific. However, not all cell types have a known specific surface marker [27].

For some cells such as cancer cells, not many cell-surface markers have been identified. In such a situation, some techniques have used a surface marker directed against surface membrane antigens that are expressed in tissue of origin, which is most often epithelial. Thus, detection of these tissue-specific surface markers in the blood stream is suggesting that cancer cells have detached from the tumor. Some techniques attempting to isolate circulating tumor cells (CTCs) from whole blood have used EpCAM, an epithelial cell adhesion molecule that is overexpressed in epithelial carcinomas such as colon and breast. However, published studies have shown inconsistent frequency of EpCAM expression in breast cancer, from as low as 35% to as high as 100% [28-31]. Thus, EpCAM cannot be considered a CTC specific marker. Additional bio-molecules such as DAPI (nucleic acid stain) and antibodies against cytokeratin (expressed on the epithelial cell membrane) and CD45 (expressed on the majority of hematopoietic cells) must be used if captured cells are to be positively identified as CTC. Therefore a cell with the phenotype EpCAM+, DAPI+, CK+, and CD45- is considered a CTC.

The number of available cells of interest poses and additional challenge; in some applications such as the isolation of CTCs from cancer patient or fetal nucleated red blood from maternal circulation, only 1-2 cell are available per milliliter (mL) of whole blood. Specificity is problematic for either method, due to the lack of a cell-specific surface marker/antibody to exclusively detect CTCs or fetal cells [32].

Use of Microfluidic Technology for Cell Separation 199

safer than "macro" platforms due to the smaller chemical quantities used and hence the lower stored energies. A microfluidic device that performs one assay is typically referred to as Lab-on-a-chip, while a device that integrates more than one step is referred to as micro

This section will briefly describe the basic concepts in the microfabrication of microfluidic devices. Microfabrication is the already subject of many textbooks and the interested reader can consult one of these for more in-depth details [33, 34]. Microfabrication is the technology developed by ICs industry to make devices and circuits with feature sizes as small as 14 nm in research, and 45 nm in production. Among these are the microprocessors and the electronic components found in computers, smart phones, television sets, and major other electronic products. Microfabrication is also used for MEMS devices (micro-electro mechanical systems), devices that include a movable part and can be used for sensing. The airbag sensor used to deploy an airbag in a vehicle, the pressure sensors inside car tires, and the electronic compass in a smart phone are all examples of MEMS devices that we unknowingly use every day. BioMEMS, microfluidic devices, and µTAS are all microfabricated devices similar to MEMS but customized for biological and chemical

Creation of a a new microfluidic device includes design of the channel(s), fluid inlet(s) and outlet(s), using a CAD (computer-aided design) software. These softwares, such as CoventorWare® [35], ANSYS CFD [36], COMSOL Multiphysics® [37], can be also used for simulation of the various design parameters such as device dimensions, heat transfer, and flow conditions, therefore narrowing the design space range in which optimum performance should be obtained. The pattern of channels is laid out with the CAD software; this is the 2D design of the device. The depth of the channel will be determined by the etching time. The drawings are transferred to a mask, typically a glass or quartz plate (transparent to UV light), covered with chrome. The chrome is etched (removed) where the UV light will expose the photoresist. The mask, similar to a stencil, transfers the pattern to the photoresist. The device is built on a substrate, which is a silicon, glass or quartz wafer, or a regular glass slide. After substrate cleaning, the photoresist, a photosensitive polymer, is applied. Photoresist is dispensed onto the substrate and it is spun at high speed (2000- 4000rpm) to create a thin (1-100 micrometer), uniform and smooth layer. The mask is placed in contact with the substrate and exposed to UV lights on a mask aligner. The photoresist is developed in a developer solution specific for it, and is removed from areas exposed to UV light (positive photoresist). The channels can be etched, with substrate material being removed from areas unprotected by the photoresist. Etching can be either wet (using chemicals) or dry (plasma etching). Deep reactive ion etching (DRIE) is a plasma etching technique typically used to achieve deep channels with vertical side walls. Use of DRIE is necessary if the etched substrate will serve as a template for molding devices in polymer such that the polymer mold can be peeled off the substrate. At this stage, the device can be

total analysis system (µTAS) [18].

applications.

**2.2. Introduction to microfabrication** 

Despite great successes, cell sorting techniques are not ideal and therefore remain an active area of research. In addition to sensitivity and specificity requirements, an ideal technique should not be overly labor intensive, should be automated and quantitative, the results should predict clinical outcome, and help the physician personalize therapeutic options. Automating sample preparation and handling would minimize human errors. Integration of preparation, cell sorting, and post processing will lead to more cost-effective instruments, and alleviate the need for trained personnel and infrastructure. Microfluidic technology enables the precise control over the cell microenvironment during separation, scales down the analyses to very small volume of blood, and has the potential for high-throughput to cell separation and analyses.
