Intact microglia are cultured and non-invasively harvested without pathological activation using a novel cultured cell recovery method
Introduction
Sophisticated tissue engineering techniques often involving cultured or engineered cells are presently required for cell transplantation devices as well as hybrid artificial organ constructs [1], [2]. For cultured cells to achieve widespread clinical use in therapies and functional replacements, however, further improvements in cell manipulation techniques such as long-term cell functional regulation and genetic manipulation must be realized. Importantly, for cell encapsulation [3], [4] and immuno-isolation methods [5], cell culture methods that provide sufficient quantities of cells with the desired differentiation and phenotypic characters must be developed. We have recently developed a novel intelligent cell culture material utilizing the well-known temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm). PIPAAm exhibits a well-described temperature transition in aqueous solution, producing reversible soluble/insoluble polymer changes near 32°C as a result of hydration/dehydration changes [6], [7]. Various temperature-sensitive polymer-based devices as well as a novel hydrophobic chromatography system have exploited this reversible aqueous polymer-phase transition [8]. PIPAAm chains covalently grafted to solid surfaces also exhibit temperature-dependent hydrophilic/hydrophobic alterations below and above 32°C. PIPAAm-grafted surfaces have therefore been recently exploited for controlling cultured cell adhesion/detachment as well as further tissue engineering applications [9], [10], [11]. At 37°C, PIPAAm-grafted surfaces in culture media exhibit a relatively hydrophobic character: plated cells readily adhere, spread and proliferate normally on this surface. By decreasing temperature below 32°C, PIPAAm-grafted surfaces rapidly begin to hydrate, and these cultured cells spontaneously begin to detach without proteolytic enzyme treatment. We have named this novel noninvasive cell harvest method as CHIP (cell harvest utilizing intelligent polymers). The CHIP method facilitates maintenance of highly differentiated cell functions for cultured primary endothelial cells and hepatocytes compared with similar cells harvested by conventional trypsin methods [12].
In the present study, we have examined highly susceptible primary brain cells—microglial cells derived from monocytes—with this noninvasive cell harvest method, CHIP. Transplantation of cells that secrete neurotrophic factors as well as neurotransmitters have been revealed to be therapeutically promising for certain brain disease treatment [4], [13]. Microglia also secrete needed neurotrophic factors. However, microglia are present in a resting state as ramified microglia in the normal matured brain, and the cells exhibit marked changes in morphology, metabolism and gene expression under pathologically activating conditions such as neurodegenerative disorders, infectious diseases [14], [15], autoimmune diseases [16], ischemia [17], inflammation [18], axotomy [19], and stab wounds [20]. One physiological role of activated microglia is to act as scavengers to remove dead cells in the brain [21]. Additional roles as antigen-presenting cells [22] and immunomodulators [19] are also suggested. Furthermore, accumulating evidence shows that microglia behave multidirectionally on surrounding neurons and glial cells by producing a wide variety of physiologically active substances including neurotrophic and cytotoxic factors [23] depending on the activated state. To date, neurotrophins, including NGF [24], BDNF [24], [25], and NT-3 [25], as well as cytokines, such as TGF-β [26], IL-3 [27], and IL-6 [28], which exert neurotrophic effects, have been shown to be expressed or produced in microglia. In addition, bFGF [29], plasminogen [30], and HGF [31] have also been identified as microglia-derived neurotrophic substances. These factors are of potential therapeutic value as they are suggested to function in neuronal growth and maturation in vivo. On the other hand, activated microglia are also suggested to be neurotoxic [32], since they also produce potentially cytotoxic molecules in pathological conditions, including reactive oxygen species [33], nitric oxide [34], glutamate [35], eicosanoids and inflammatory cytokines [36]. Furthermore, activated microglia are also implicated in influencing the proliferation of astrocytes, the differentiation of oligodendrocytes, and angiogenesis through the secretion of IL-1 [37], PDGF [38], and TNF-α [39], respectively. The ability to culture these cells on a large scale and precisely regulate “microglial activation” should both prove essential in successful cell transplantation therapies for brain diseases. In this context, unique, new advantages of the CHIP method for microglial culture are compared with the conventional trypsin cell harvest method.
Section snippets
Preparation of temperature-responsive cell culture surfaces grafted with poly(N-isopropylacrylamide) (PIPAAm)
PIPAAm-grafted cell culture dishes were prepared as described previously [40]. In brief, 70 μl of 60% (w/w) N-isopropylacrylamide (KOHJIN Co.) solution dissolved in 2-propanol was added to each Falcon 3002 dish (60 mm diameter) and then irradiated with a 0.3 MGy electron beam using an area beam electron processing system (Nissin-High Voltage Co. Ltd.). The reaction facilitates the covalent grafting of PIPAAm polymers to cell culture surfaces. Homogeneous coverage of PIPAAm-grafted dishes was
Results
PIPAAm was covalently grafted to the surfaces of commercial cell culture dishes. The molecular structure for PIPAAm is shown in Fig. 1a. PIPAAm exhibits a dehydrated hydrophobic, compact chain conformation above 32°C, while below 32°C, the polymer spontaneously hydrates, becoming hydrophilic, with an extended chain conformation. When cells are plated on PIPAAm-grafted dishes at 37°C, they attach, spread and grow on the polymer-grafted surface. By reducing temperature, cell surface detachment
Discussion
As an alternative to the conventional trypsin method, the CHIP method introduced here has been shown to exhibit advantages in terms of simplicity and convenience, cell recovery efficiencies, and its non-invasiveness. In contrast, trypsin method was noted to be inadequate for detaching microglia because the treatment altered their metabolism and cellular responses. Two possible reasons for these alterations are considered. One possibility is that trypsin method may select for certain
Acknowledgements
We are thankful to Professor David W. Grainger of Colorado State University, for his valuable comments. This study was supported by Grants from the Ministry of Health and Welfare of Japan, a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan, Grants from the Japan Science and Technology Agency, and the Japan Society for the Promotion of Science, “Research for the Future” Program (JSPS-RFTF96I00201).
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