This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.
To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 µm diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.
Cell transplantation therapy has attracted widespread attention for treating various intractable diseases. The activity and half-life of bioactive factors that are secreted by the transplanted cells are essential for improved therapeutic effectiveness of a cell transplantation system. Genetic modification of the cells prior to transplantation is a beneficial technique to regulate and manipulate cellular functions, including the secretion of the bioactive factors. It is also important to maintain a favorable microenvironment for the cells for avoiding cell death or loss of cell activity. Three-dimensional (3D) spheroid cell culture, in which cell-to-cell interactions are well preserved, is promising for this purpose, for example, for improving albumin secretion from primary hepatocytes and promoting multi-lineage differentiation from mesenchymal stem cells (MSCs) 1-7.
In this study, a novel combination system of spheroid culture and gene transfection is used to serve as a platform for genetically modified cell transplantation. For creating spheroid cells, a spheroid culture system on micropatterned culture plates is used. On these plates, cell adhesion areas of 100 µm diameter are regularly arrayed in a two-dimensional manner and are surrounded by non-adhesive areas coated by a PEG matrix3. By seeding an adequate number of cells, arrays of 3D spheroids of 100 µm in diameter are formed corresponding to the micropatterned culture bed.
The spheroids are recovered without disrupting their 3D structure by using thermosensitive cell-culture plates, that were coated with a thermosensitive polymer, poly(iso-propylacrylamide) (PIPAAm) 8-10. The micropatterned architecture is constructed on the thermosensitive plates (custom-built). By simply lowering the temperature of the plates, the spheroids are detached from the culture bed and dispersed in phosphate buffered saline (PBS). Thus, a large number of spheroids with a uniform size of 100 µm can be obtained in the form of an injectable suspension.
Figure 1. Schematic representation of the spheroid culture system on a micropatterned plate. Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle. It is composed of plasmid DNA (pDNA) and polyethylene glycol (PEG)-polycation block copolymers11. These have a characteristic core-shell structure consisting of a PEG shell and an inner core of condensed pDNA, allowing safe and effective gene introduction into cells for therapeutic purposes11. Please click here to view a larger version of this figure.
Figure 2. Structure of the polyplex nanomicelle formed by the complex of nucleic acids and PEG-block-polycation block copolymers. In this study, the primary advantage of this technique is that the spheroid structure is not disrupted during gene transfection by the nanomicelles. After nanomicelle-mediated transfections of rat primary hepatocyte spheroids, prolonged transgene expression is obtained for more than a month with continuous albumin secretion from the hepatocytes at a level comparable to that of untransfected spheroids12. The transgene expression and albumin secretion from the spheroids are also maintained after recovery from the thermosensitive plates. It is evident that nanomicelles can safely facilitate gene introduction without impairing the innate functions of the hepatocytes. Thus, the combination of spheroid cells cultured on thermosensitive micropatterned plates with gene introduction using nanomicelles is a promising platform for genetically modified cell transplantation. Please click here to view a larger version of this figure.
All the animal studies were conducted with the approval of the Animal Care and Use Committee of the University of Tokyo, Tokyo, Japan.
1. Cell Preparation
2. Preparation of 3D Cell Spheroids
3. Preparation of Polyplex Nanomicelles
4. Gene Transfection into Spheroids
5. Recovery and Transplantation of Cell Spheroids
6. Evaluation of Transgene Expression
Gene transfection of the Gaussia luciferase-expressing pDNA was performed in the spheroids formed by the hepatocytes or MSCs using polyplex nanomicelles or the control lipid-based transfection reagent12. The nanomicelles induced almost no change in the spheroid structure compared with non-transfected spheroids on the micropatterned plates, whereas the control reagent significantly disrupted the structure a day after the transfection (Figure 3). After transfection using the nanomicelles, consistent albumin secretion and the transgene (Gaussia luciferase) expression were well maintained for almost a month. However, when the control reagent was used, no albumin secretion was observed, although the degree of the transgene expression was similar to that obtained from the nanomicelles (Figure 4) 12.
During spheroid recovery using the thermosensitive micropatterned plates, the 3D structure of the spheroids was well preserved for both the primary hepatocytes and the MSCs in suspension after recovery from the cooled plates (Figure 5). The structure was also well maintained after passing the spheroids through injection needles with a sufficiently large caliber, such as the 23 (400 µm) and 27 G (220 µm) needles.
After subcutaneous injections of the recovered hepatocyte spheroids, luciferase expression was detected in the animals for more than a few weeks (Figure 6) 20. Albumin expression from transplanted hepatocytes was observed in the host tissue20, suggesting that the cell function was preserved through the process of spheroid recovery and transplantation. To test their potential for therapeutic applications, the hepatocyte spheroids were also transfected with erythropoietin-encoding pDNA. After subcutaneous transplantation of the spheroids expressing erythropoietin, a significant hematopoietic effect was obtained in the animals for one month (Figure 7) 20.
Figure 3. Microscopic images of hepatocyte spheroids (A, B) and MSC spheroids (C, D) on a micropatterned plate a day after gene transfection, using polyplex nanomicelles (A, C) or lipid-based reagent (B, D). Scale bar: 100 µm. Please click here to view a larger version of this figure.
Figure 4. (A) Transgene expression of Gaussia luciferase after transfecting the hepatocyte spheroids. (B) Albumin secretion from the hepatocyte spheroids or hepatocytes in a monolayer culture after the transfection. Spheroids were transfected using nanomicelle (●) or control lipid-based transfection reagent (○), or naked pDNA (▲). The albumin secretion from the control (untransfected) hepatocytes (in spheroids (△) or monolayer culture (×)) is also shown. The results are represented as mean ± SEM; n = 4 for monolayer culture and n = 6 for spheroids. (Reprinted with permission from reference 12). Please click here to view a larger version of this figure.
Figure 5. Microscopic images of the hepatocyte spheroids in suspension after passing them through (A) 23- or (B) 27 G injection needles. Scale bar: 100 µm. Please click here to view a larger version of this figure.
Figure 6. Luciferase expression in host mice after hepatocyte transplantation. After 24 hrs after transfection with luciferase (GL4)-encoding pDNA, the hepatocyte spheroids and single-cell suspension from the monolayer cultures were transplanted into the subcutaneous tissue of the abdominal region. The luciferase expression in the host mice was evaluated using an IVIS Imaging System (Reprinted with permission from reference 20).Please click here to view a larger version of this figure.
Figure 7. Hematopoiesis after transplantation of the erythropoietin-expressing hepatocytes. Hepatocytes in the spheroid and monolayer cultures were transfected with erythropoietin-encoding pDNA. After 24 hrs of transfection, the spheroids and single-cell suspensions from the monolayer cultures were subcutaneously transplanted into the mouse abdomen. At 22 and 28 days after transplantation, (A) hemoglobin and (B) hematocrit levels were measured from the blood samples. Data are presented as the mean ± SEM; n = 12 for spheroid and monolayer groups and n = 6 for untreated controls. Statistical significance was determined by a 2-tailed Student's t-test (Reprinted with permission from reference 20). Please click here to view a larger version of this figure.
In this protocol, it is critical to maintain the 3D structure of spheroids during the steps of gene introduction and spheroid recovery. It is essential to maintain a favorable microenvironments for the cells to avoid cell death or loss of cell activity. For example, albumin secretion, a representative innate function of hepatocytes, is well preserved in the hepatocyte spheroids, while the hepatocytes in the conventional monolayer culture rapidly lose their secretory capacity a few days after seeding12. For MSCs, the spheroids significantly enhance their efficiency of differentiation into adipocytes or osteoblasts, with increased expression levels of genes involved in adipogenesis and osteogenesis, and down-regulation of genes that maintain the MSC self-renewal phenotype4.
For gene introduction, various transfection reagents had been tested previously, including lipid-based ones (Figure 3, 4) and polymer-based ones, such as polyethylenimine. Unfortunately, none of the reagents provided successful gene introduction without disrupting the spheroid structure. Especially, the lipid-based reagents tend to induce complete disruption of the spheroids, although the transgene expression is efficient, even in the remaining cells on the micropatterned plates. It is likely that the lipid-based reagents induce significant membrane destabilization, leading to high transgene expression, but disrupt the spheroids by rendering the cell-to-cell interactions unstable.
In contrast, the cationic polymer disrupted the spheroids to a lesser extent than the lipids, especially in the conditions of low N/P ratios, to form the polyplexes. The standard protocol shown here uses polyplex nanomicelles that possess PEG shielding. Alternatively, polyplexes with cationic homopolymers, such as polyethylenimine, can also be used for gene introduction into the spheroids. Indeed, cationic polyplexes without the PEG surface generally provide high transgene expression in in vitro transfections, and are attractive options depending on the cell type and purpose of the spheroid transplantation.
A characteristic feature of this protocol is the use of micropatterned culture plates coated with a thermosensitive polymer. The micropatterned arrays can produce a large number of cell spheroids with a uniform diameter. This protocol uses 100 µm diameter arrays, but this aspect is flexible; however, very large diameters, such as 500 µm, often cause cell necrosis in the core of the spheroids (unpublished data). By simply cooling the plates on ice, the spheroids can be obtained in the form of an injectable suspension by gentle aspiration with a syringe. A potential limitation of this technique is the size of the needle used to inject the spheroids. It was confirmed that a 27-G needle can be safely used for spheroids with 100 µm diameter. However, narrow needles cannot be applied for these spheroids. Since a 27-G needle is too large, especially for transplanting spheroids into mouse spinal cords, a possible alternative is to use scaffolds to retain cells inside them.
This protocol can be potentially applied for several purposes of cell transplantation. Compared to the suspension spheroid culture system, the micropatterned plates have the advantage that a sufficient number of spheroids with uniform diameter can be easily obtained. Although the method of gene introduction can be flexible, as mentioned previously, polyplex nanomicelles with PEG shielding are particularly effective for avoiding spheroid disruption. Hopefully, this system will improve the therapeutic effects of transplanted cells.
The authors have nothing to disclose.
We deeply appreciate Dr. Takeshi Ikeya and technical staff in Toyo Gosei, Tokyo, Japan for providing thermosensitive micropatterned culture plates as well as scientific advice. We also thank Ms. Satomi Ogura, Ms. Sae Suzuki, Ms. Asuka Miyoshi and Ms. Katsue Morii for technical assistance with animal experiments. This work was financially supported in part by the JSPS KAKENHI Grant-in-Aid for Scientific Research, the Center of Innovation (COI) Program and the S- innovation program from the Japan Science and Technology Agency (JST), and the JSPS Core-to-Core Program, A. Advanced Research Networks.
Pen-Strep-Glut | GIBCO | ||
Dexamethasone | Wako Pure Chemical Industries | 041-18861 | |
Nicotinamide | Wako Pure Chemical Industries | 141-01202 | |
Hank’s buffered salt and L-ascorbic acid 2-phosphate (Asc-2P) | Sigma-Aldrich | A8960 | |
Human epidermal growth factor (hEGF) | Toyobo | PT10015 | |
Cell-able multi-well plates | Toyo Gosei | PP-12 | |
Thermosensitive cell culture plates (Upcell) | CellSeed Inc | The micropatterned architecture is constructed on the thermosensitive plates (custom-built by Toyo Gosei) | |
Lipid-based transfection reagent (FuGENE HD) | Promega | E2311 | |
Renilla Luciferase Assay System | Promega | E2810 | |
pGL4 Luciferase Reporter Vector | Promega | E6651 | |
pDNA expressing Gaussia luciferase | New England BioLabs | N8082S | |
Mouse erhthropoietin-expressing vector | Origene | MC208445 | |
pCAG-GS | Kindly provided by Laboratory for Pluripotent Cell Studies, Center for Developmental Biology, RIKEN | ||
Escherichia coli DH5α competent cells | Takara | 9057 | |
Endotoxin-free plasmid DNA purification system | Nippon Genetics | NucleoBond Xtra EF | |
collagenase | Wako Pure Chemical Industries | 639-00951 | |
trypsin inhibitor | GIBCO | R-007-100 | |
Luminometer | Promega | GloMax™ 96 Microplate Luminometer | |
IVIS Imaging System | Xenogen Corp. | Xenogen IVIS Spectrum in vivo imaging system | |
blood sample analyzer | Sysmex | pocH-100i Automated Hematology Analyzer |