基于液滴微流控的细胞凝胶微球研究进展

液滴微流控技术在微纳米尺度上对多种流体的流动进行精确控制,从而能够以高通量的方式生成结构可调和成分可控的微纳米液滴。通过结合合适的水凝胶材料和制造方法,可以将单个或多个细胞高效地封装进水凝胶中,制备细胞凝胶微球。细胞凝胶微球可以为细胞的增殖、分化等提供一个三维的、相对独立可控的微环境,在三维细胞培养、组织工程与再生医学、干细胞研究和单细胞研究等生命科学领域具有重要价值。本文主要综述了基于液滴微流控技术的细胞凝胶微球的制备及其在生物医学领域的应用,并对未来的研究工作提出了展望。     

ODN 491, a novel antisense oligodeoxynucleotide that targets thymidylate synthase, exerts cell-specific effects in human tumor cell lines

Thymidylate synthase (TS) is essential for DNA replication and is a target for cancer chemotherapy. However, toxicity to normal cells and tumor cell drug resistance necessitate development of new therapeutic strategies. One such strategy is to use antisense (AS) technology to reduce TS mRNA and protein levels in treated cells. We have developed oligodeoxynucleotides (ODNs) that target different regions of TS mRNA, inhibit human tumor cell proliferation as single agents, and enhance cytotoxicity of clinically useful TS protein-targeting drugs. Here we describe ODN 491, a novel 20mer AS ODN complementary to a previously untargeted portion of the TS mRNA coding region. AS ODN 491 decreased TS mRNA levels to different degrees in a panel of human tumor-derived cell lines, and induced different physiological effects in a tumor cell line-dependent manner. ODN 491 (like AS TS ODN 83, previously shown to be effective) decreased TS protein levels in HeLa cells with a concomitant increase in sensitivity to TS-targeting chemotherapeutics. However (and contrary to HeLa cell response to an AS ODN 83), it did not, as a single agent, inhibit HeLa cell proliferation. In MCF-7 cells, ODN 491 treatment was less effective at reducing TS mRNA and did not reduce TS protein, nor did it enhance sensitivity to TS-targeting or other chemotherapeutics. Moreover, specifically in MCF-7 cells but not HeLa cells, ODN 491 as a single agent induced apoptosis. These data indicate that AS TS ODN 491 is an effective AS reagent targeting a novel TS mRNA region. However, treatment of tumor cell lines with AS TS ODNs targeting different TS mRNA regions results in a pattern of physiological effects that varies in a tumor cell line-specific fashion. In addition, the capacity of different AS TS ODNs to induce physiological effects does not correlate well with their capacity to reduce TS mRNA and/or protein and, further, depends on the region of TS mRNA selected for targeting. Recognition of tumor cell-specific and mRNA region-specific variability in response to AS TS ODNs will be important in designing AS TS ODNs for potential clinical use.     

AAV-mediated gene targeting methods for human cells

Gene targeting with adeno-associated virus (AAV) vectors has been demonstrated in multiple human cell types, with targeting frequencies ranging from 10(-5) to 10(-2) per infected cell. These targeting frequencies are 1-4 logs higher than those obtained by conventional transfection or electroporation approaches. A wide variety of different types of mutations can be introduced into chromosomal loci with high fidelity and without genotoxicity. Here we provide a detailed protocol for gene targeting in human cells with AAV vectors. We describe methods for vector design, stock preparation and titration. Optimized transduction protocols are provided for human pluripotent stem cells, mesenchymal stem cells, fibroblasts and transformed cell lines, as well as a method for identifying targeted clones by Southern blots. This protocol (from vector design through a single round of targeting and screening) can be completed in ～10 weeks; each subsequent round of targeting and screening should take an additional 7 weeks.     

Microselection – affinity selecting antibodies against a single rare cell in a heterogeneous population

Rare cells not normally present in the peripheral bloodstream, such as circulating tumour cells, have potential applications for development of non‐invasive methods for diagnostics or follow up. Obtaining these cells however require some means of discrimination, achievable by cell type specific antibodies. Here we have generated a microselection method allowing antibody selection, by phage display, targeting a single cell in a heterogeneous population. One K562 cell (female origin) was positioned on glass slide among millions of lymphocytes from male donor, identifying the K562 cell by FISH (XX). Several single cell selections were performed on such individual slides. The phage particles bound to the target cell is protected by a minute disc, while inactivating all remaining phage by UV‐irradiation; leaving only the phage bound to the target cell viable. We hereby retrieved up to eight antibodies per single cell selection, including three highly K562 cell type specific.     

Correlation between cell survival,clonogenic activity and micronuclei induction in DMBA-OC-1R cells treated with immunospecific albumin microspheres containing cisplatin and 5-fluorouracil

An ideal chemotherapeutic strategy would be to deliver a high concentration of drug that would be released in sustained small amounts from targeted microspheres to effectively kill only the tumour cells and thus reduce toxicity to normal tissue. Clonogenic and cell survival growth curve assays, as well as the micronucleus assay, were used to determine the feasibility of employing targeted immunomicrospheres in the treatment of cancer. Cells of a rodent ovarian carcinoma cell line, were exposed to cisplatin and 5-fluorouracil, either as free drug or encapsulated in albumin microspheres that were either conjugated to monoclonal antibodies or not. In cell survival growth curve assays, cell survival was reduced to 1.2% of the control when cells were treated with drug-containing immunomicrospheres. 3.2-fold more micronuclei were found in those cells that had been exposed to the drugs in immunomicrospheres than in those subjected to untargeted microspheres. All three assays demonstrated that the targeted immunomicrospheres were more effective in delivering cisplatin and 5-fluorouracil directly to the cells than the unconjugated microspheres, thus suggesting that targeted chemotherapy might be a more effective option in the treatment of cancer.     

Effect of cytochalasin B, nocodazole and irradiation on migration and internalization of cells and microspheres in tumor cell spheroids

The effect of cytochalasin B (CB), nocodazole, and irradiation on the adherence and internalization of 3H-labeled EMT6 spheroid-derived single cells and inert microspheres in unlabeled, intact EMT6 multicellular spheroids has been examined. CB inhibited adhesion and internal migration, whereas nocodazole did not stop adhesion but did prevent later internalization. Treatment of labeled cells with 5, 15 and 25 Gy 250 kV X-rays before adherence did not effect their adherence or later internalization. The same radiation treatments administered to the spheroids either immediately before or after the introduction of unirradiated single cells did not affect adherence, but the depths reached by labeled cells and microspheres were reduced largely because of the consequent reduction in spheroid growth. Microsphere size (9, 15, or 25 microns) and surface charge (negative, or non-ionic) had minimal, if any, effect on the adherence and internalization of these particles.     

Delivery of Bioactive Lipids from Composite Microgel-Microsphere Injectable Scaffolds Enhances Stem Cell Recruitment and Skeletal Repair

In this study, a microgel composed of chitosan and inorganic phosphates was used to deliver poly(lactic-co-glycolic acid) (PLAGA) microspheres loaded with sphingolipid growth factor FTY720 to critical size cranial defects in Sprague Dawley rats. We show that sustained release of FTY720 from injected microspheres used alone or in combination with recombinant human bone morphogenic protein-2 (rhBMP2) improves defect vascularization and bone formation in the presence and absence of rhBMP2 as evaluated by quantitative microCT and histological measurements. Moreover, sustained delivery of FTY720 from PLAGA and local targeting of sphingosine 1-phosphate (S1P) receptors reduces CD45+ inflammatory cell infiltration, promotes endogenous recruitment of CD29+CD90+ bone progenitor cells and enhances the efficacy of rhBMP2 from chitosan microgels. Companion in vitro studies suggest that selective activation of sphingosine receptor subtype-3 (S1P₃) via FTY720 treatment induces smad-1 phosphorylation in bone-marrow stromal cells. Additionally, FTY720 enhances stromal cell-derived factor-1 (SDF-1) mediated chemotaxis of CD90+CD11B-CD45- bone progenitor cells in vitro after stimulation with rhBMP2. We believe that use of such small molecule delivery formulations to recruit endogenous bone progenitors may be an attractive alternative to exogenous cell-based therapy.     

T cell apoptosis and induction of foxp3+ regulatory T cells underlie the therapeutic efficacy of CD4 blockade in experimental autoimmune encephalomyelitis

The pathogenesis of multiple sclerosis requires the participation of effector neuroantigen-specific T cells. Thus, T cell targeting has been proposed as a promising therapeutic strategy. However, the mechanism underlying effective disease prevention following T cell targeting remains incompletely known. We found, using several TCR-transgenic strains, that CD4 blockade is effective in preventing experimental autoimmune encephalopathy and in treating mice after the disease onset. The mechanism does not rely on direct T cell depletion, but the anti-CD4 mAb prevents the proliferation of naive neuroantigen-specific T cells, as well as acquisition of effector Th1 and Th17 phenotypes. Simultaneously, the mAb favors peripheral conversion of Foxp3(+) regulatory T cells. Pre-existing effector cells, or neuroantigen-specific cells that undergo cell division despite the presence of anti-CD4, are committed to apoptosis. Therefore, protection from experimental autoimmune encephalopathy relies on a combination of dominant mechanisms grounded on regulatory T cell induction and recessive mechanisms based on apoptosis of neuropathogenic cells. We anticipate that the same mechanisms may be implicated in other T cell-mediated autoimmune diseases that can be treated or prevented with Abs targeting T cell molecules, such as CD4 or CD3.     

A Simple Method for Encapsulating Single Cells in Alginate Microspheres Allows for Direct PCR and Whole Genome Amplification

Microdroplets are an effective platform for segregating individual cells and amplifying DNA. However, a key challenge is to recover the contents of individual droplets for downstream analysis. This paper offers a method for embedding cells in alginate microspheres and performing multiple serial operations on the isolated cells. Rhodobacter sphaeroides cells were diluted in alginate polymer and sprayed into microdroplets using a fingertip aerosol sprayer. The encapsulated cells were lysed and subjected either to conventional PCR, or whole genome amplification using either multiple displacement amplification (MDA) or a two-step PCR protocol. Microscopic examination after PCR showed that the lumen of the occupied microspheres contained fluorescently stained DNA product, but multiple displacement amplification with phi29 produced only a small number of polymerase colonies. The 2-step WGA protocol was successful in generating fluorescent material, and quantitative PCR from DNA extracted from aliquots of microspheres suggested that the copy number inside the microspheres was amplified up to 3 orders of magnitude. Microspheres containing fluorescent material were sorted by a dilution series and screened with a fluorescent plate reader to identify single microspheres. The DNA was extracted from individual isolates, re-amplified with full-length sequencing adapters, and then a single isolate was sequenced using the Illumina MiSeq platform. After filtering the reads, the only sequences that collectively matched a genome in the NCBI nucleotide database belonged to R. sphaeroides. This demonstrated that sequencing-ready DNA could be generated from the contents of a single microsphere without culturing. However, the 2-step WGA strategy showed limitations in terms of low genome coverage and an uneven frequency distribution of reads across the genome. This paper offers a simple method for embedding cells in alginate microspheres and performing PCR on isolated cells in common bulk reactions, although further work must be done to improve the amplification coverage of single genomes.     

Cell deposition system based on laser guidance

We have designed a laser cell deposition system that employs the phenomenon of laser guidance to place single cells at specific points in a variety of in vitro environments. Here, we describe the components of the system: the laser optics, the deposition chamber, the microinjection cell feeding system and our custom system control software application. We discuss the requirements and challenges involved in laser guidance of cells and how our present system overcomes these challenges. We demonstrate that the patterning system is accurate within one micrometer by repeatedly depositing polymer microspheres and measuring their position. We demonstrate its ability to create highly defined living patterns of cells by creating a defined pattern of neurons with neurite extensions displaying normal function. We found that the positional accuracy of our system is smaller than the variations in cell size and pattern disruptions that occur from normal cell movement during substrate adhesion. The laser cell deposition system is a potentially useful tool that can be used to achieve site- and time-specific placement of an individual cell in a cell culture for the systematic investigation of cell-cell and cell-extracellular matrix interactions.     

Localized delivery of epidermal growth factor improves the survival of transplanted hepatocytes

Hepatocyte transplantation may provide a new approach for treating a variety of liver diseases if a sufficient number of the transplanted cells survive over an extended time period. In this report, we describe a technique to deliver growth factors to transplanted hepatocytes to improve their engraftment. Epidermal growth factor (EGF) was incorporated (0.11%) into microspheres (19 +/- 12 mum) fabricated from a copolymer of lactic and glycolic acid using a double emulsion technique. The incorporated EGF was steadily released over 1 month in vitro, and it remained biologically active, as determined by its ability to stimulate DNA synthesis, cell division, and long-term survival of cultured hepatocytes. EGF-containing microspheres were mixed with a suspension of hepatocytes, seeded onto porous sponges, and implanted into the mesentery of two groups of Lewis rats. The first group of animals had their portal vein shunted to the inferior vena cava prior to cell transplantation (portal-caval shunt = PCS), and the second group of animals did not (non-PCS). This surgical procedure improves the survival of transplanted hepatocytes. The engraftment of transplanted hepatocytes in PCS animals was increased two-fold by adding EGF microspheres, as compared to adding control microspheres that contained no growth factors. Devices implanted into non-PCS animals had fewer engrafted hepatocytes than devices implanted into PCS animals, regardless of whether blank or EGF-containing microspheres were added. These results first indicate that it is possible to design systems which can alter the microenvironment of transplanted hepatocytes to improve their engraftment. They also suggest that hepatocyte engraftment is not improved by providing single growth factors unless the correct environment (PCS) is provided for the transplanted cells. (c) 1996 John Wiley & Sons, Inc.     

Focal adhesion targeting of v-Crk is essential for FAK phosphorylation and cell migration in mouse embryo fibroblasts deficient src family kinases or p130CAS

We examined the consequences of v-Crk expression in mouse embryo fibroblasts deficient Src family kinases or p130CAS. We found that Src kinases are essential for p130CAS/v-Crk signaling leading to FAK phosphorylation and cell migration in which Src is likely to mediate the focal adhesion targeting of v-Crk. SYF cells showed only low levels of FAK phosphorylation and cell migration, even in the presence of v-Crk. Expression of v-Crk restored migration of p130CAS-deficient cells to the level of wild-type cells, most likely through the targeting of v-Crk to focal adhesions by cSrc. In addition, we identified a new v-Crk-interacting protein that mediates v-Crk signaling in p130CAS-deficient cells. Using RT-PCR and caspase cleavage assays, we confirmed that this protein is not p130CAS and is responsible for maintaining v-Crk/Src signaling and migration in these. These findings suggest that focal adhesion targeting of v-Crk is essential in v-Crk-mediated cellular signaling and that v-Crk must form a complex with p130CAS or a p130CAS substitute to transduce signaling from the extracellular matrix.     

Three-dimensional environment renders cancer cells profoundly less susceptible to a single amino acid starvation

Increased amino acid requirement of malignant cells is exploited in metabolic antitumor therapy, e.g., enzymotherapies based on arginine or methionine deprivation. However, studies on animal models and clinical trials revealed that solid tumors are much less susceptible to single amino acid starvation than could be expected from the in vitro data. We conducted a comparative analysis of the response of several tumor cell lines to single amino acid starvation in 2-D monolayer versus 3-D spheroid culture. We revealed for the first time that in comparison with monolayer culture tumor cells, spheroids are much less susceptible to the deprivation of individual amino acids (i.e., arginine, leucine, lysine or methionine). Accordingly, even after prolonged (up to 10 days) starvation, spheroid cells could readily resume proliferation when appropriate amino acid was resupplemented. In the case of arginine deprivation, similar apoptosis induction was detected both in 2-D and 3-D culture, suggesting that this process does not determine the level of tumor cell sensitivity to this kind of treatment. It was also observed that spheroids much better mimic the in vivo ability of tumor cells to utilize citrulline as arginine precursor for growth in amino acid deficient environment. We conclude that 3-D spheroid culture better reflects in vivo tumor cell response to single amino acid starvation than 2-D monolayer culture and should be used as an integral model in the studies of this type of antitumor metabolic targeting.     

Sampling efficiency of a single-cell capillary electrophoresis system.

Capillary electrophoresis (CE) combined with a laser-induced fluorescence (LIF) detection scheme is a powerful approach for single-cell analysis. For measurements requiring a high temporal resolution, CE-LIF is often combined with cell lysis systems based on pulsed lasers. Although extremely rapid, laser lysis has raised some concerns about the efficiency at which the cell contents are sampled. We have assembled a single-cell CE-LIF mounted on the stage of a microscope. This system was coupled with a nanosecond pulsed laser for cell lysis. We have analyzed green fluorescent protein (GFP) expressed in single mammalian cells and developed a novel approach to estimate the cell sampling efficiency (SE) based on the use of fluorescent calibration microspheres and flow cytometry. A significant advantage of this method is that it does not require any knowledge or assumption regarding the cell volume. We have evaluated the SE for different laser pulse energies (from 2 to 9 microJ) and two different pulse focal positions in the xy plane (0-10 microm from the center of the cell). We found the maximum SE at the lowest energy (2 microJ), with the pulse focused directly on the cell. We have demonstrated the utility of a novel method to measure the SE of a single-cell CE system. The measurements presented in this study indicate that rapid cell lysis with nanosecond lasers requires careful optimization of pulse parameters for maximum sampling of the cell contents.     

Nanoparticles,molecular biosensors,and multispectral confocal microscopy

Complex, multilayered nanoparticles hold great promise for more sophisticated drug/gene delivery systems to single cells. Outermost layers can include cell targeting and cell-entry facilitating molecules. The next layer can include intracellular targeting molecules for precise delivery of the nanoparticle complex inside the cell of interest. Molecular biosensors can be used to confirm the presence of expected molecules (for example, reactive oxygen species (ROS) as a surrogate molecule for signs of infection, or for activation in radiation damage, etc.) prior to delivery of counter-measure molecules such as drugs or gene therapy. They can also be used as a feedback control mechanism to control the proper amount of drug/gene delivery for each cell. Importantly, the full nanoparticle system can be used to prevent any cells from encountering the drug unless that cell is specifically targeted. Thus, if a cell is initially non-specifically targeted, a secondary check for other molecular targets which must also be present inside the target cell of interest can be used to catch initial targeting mistakes and prevent subsequent delivery of treatment molecules to the wrong cells. The precise intracellular location of nanoparticles within specific regions of a cell can be confirmed by 3D multispectral confocal microscopy. These single cell molecular morphology measurements can be extended from individual cells, to other cells in a tissue in tissue monolayers or tissue sections.     

<Emphasis Type="Italic">Bellis perennis</Emphasis>: a useful tool for protein localization studies

Fluorescent fusion proteins together with transient transformation techniques are commonly used to investigate intracellular protein localisation in vivo. Biolistic transfection is reliable, efficient and avoids experimental problems associated with producing and handling fragile protoplasts. Onion epidermis pavement cells are frequently used with this technique, their excellent properties for microscopy resulting from their easy removal from the underlying tissues and large size. They also have advantages over mesophyll cells for fluorescence microscopy, as they are devoid of chloroplasts whose autofluorescence can pose problems. The arrested plastid development is peculiar to epidermal cells, however, and stands in the way of studies on protein targeting to plastids. We have developed a system enabling studies of in vivo protein targeting to organelles including chloroplasts within a photosynthetically active plant cell with excellent optical properties using a transient transformation procedure. We established biolistic transfection in epidermal pavement cells of the lawn daisy (Bellis perennis L., cultivar “Galaxy red”) which unusually contain a moderate number of functional chloroplasts. These cells are excellent objects for fluorescence microscopy using current reporters, combining the advantages of the ease of biolistic transfection, the excellent optical properties of a single cell layer and access to chloroplast protein targeting. We demonstrate chloroplast targeting of plastid-localised heme oxygenase, and two further proteins whose localisation was equivocal. We also demonstrate unambiguous targeting to mitochondria, peroxisomes and nuclei. We thus propose that the Bellis system represents a valuable tool for protein localisation studies in living plant cells.     

Single-cell electroporation for gene transfer in vivo

We report an electroporation technique for targeting gene transfer to individual cells in intact tissue. Electrical stimulation through a micropipette filled with DNA or other macromolecules electroporates a single cell at the tip of the micropipette. Electroporation of a plasmid encoding enhanced green fluorescent protein (GFP) into the brain of intact Xenopus tadpoles or rat hippocampal slices resulted in GFP expression in single neurons and glia. In vivo imaging showed morphologies, dendritic arbor dynamics, and growth rates characteristic of healthy cells. Coelectroporation of two plasmids resulted in expression of both proteins, while electroporation of fluorescent dextrans allowed direct visualization of transfer of molecules into cells. This technique will allow unprecedented spatial and temporal control over gene delivery and protein expression.     

Lipid microspheres incorporated by U937 cells via their specific receptors.

Lipid microspheres (LM), currently in clinical use as drug carriers, mainly consist of soybean oil as a core and lecithin as a surfactant. The purpose of our study wass to determine whether or not LM incorporation is receptor-mediated. U937 cells resuspended in a serum-free medium abundantly took up unmodulated LM. A binding study showed that U937 cells had a single binding site for LM (410 sites/cell at 24 degrees C; 100 sites/cell at 4 degrees C). Inhibition assays revealed that lecithin liposome, lysophosphatidylcholines, activated alpha2-macroglobulin, and HDL did not affect the binding of LM to U937 cells. VLDL strongly, and LDL and AcLDL moderately, inhibited the binding of LM to U937 cells. Ligand blotting analysis revealed that unmodulated LM in an apoprotein-free buffer directly bound to a 40 kDa protein in the cell membrane fraction. These results suggest that LM that is not modulated by any protein is incorporated by specific cells via receptor-mediated processes.     

High-Fidelity Correction of Mutations at Multiple Chromosomal Positions by Adeno-Associated Virus Vectors

The gene targeting techniques used to modify chromosomes in mouse embryonic stem cells have had limited success with many other cell types, especially normal primary cells with restricted growth capacity outside the organism. This is due in large part to the technical problems and/or inefficiency of conventional DNA transfer methods, as well as the low rates of homologous recombination obtained in unselected cell populations. We recently described an alternative approach in which adeno-associated virus (AAV) vectors were used to modify homologous chromosomal sequences, and targeting rates close to 1% were observed at the single copy hypoxanthine phosphoribosyl transferase (HPRT) locus in normal human cells (D. W. Russell and R. K. Hirata, Nat. Genet. 18:325-330, 1998). Here we report experiments in which we used a retroviral shuttle vector system to introduce and characterize target loci in human chromosomes, and demonstrate that AAV vectors can correct several types of mutations with high fidelity, independent of chromosomal position. The gene targeting rates varied depending on the type of mutation being corrected, implicating cellular mismatch recognition functions in the reaction. Since AAV vectors can efficiently deliver DNA to many cell types both in vivo and ex vivo, our results suggest that AAV-mediated gene targeting will have wide applicability, including therapeutic gene correction.