Efficient gene delivery into cell lines and stem cells using baculovirus

Abstract

Baculovirus is a promising vector for transducing numerous types of mammalian cells. We have developed hybrid baculovirus vectors and protocols for the efficient transduction of a variety of cell lines, primary cells and stem cells, including bone marrow–derived mesenchymal stem cells (BMSCs) and adipose-derived stem cells (ASCs). The hybrid vector enables intracellular minicircle formation and prolongs transgene expression. The advantages of this transduction protocol are that baculovirus supernatant alone needs to be added to cells growing in medium, and transduction occurs after only 4–6 h of incubation at room temperature (25 °C) with gentle shaking. The entire procedure, from virus generation to transduction, can be completed within 4 weeks. Compared with other transduction procedures, this protocol is simple and can confer efficiencies >95% for many cell types. This protocol has potential applications in tissue regeneration, as transduced cells continue to express transgenes after implantation. For example, transduction of rabbit ASCs (rASCs) with growth factor–encoding hybrid baculovirus vectors, as described as an example application in this protocol, enables robust and sustained growth factor expression, stimulates stem cell differentiation and augments tissue regeneration after implantation.

Introduction

Baculovirus (Autographa californica multiple nucleopolyhedrovirus, AcMNPV) is an insect enveloped virus and is highly infectious to cultured insect cells, and thus baculovirus is often genetically engineered to infect host insect cells for recombinant protein production. Since it was found that baculovirus is able to transduce mammalian cells^1,2, numerous permissive cells of human, rodent, porcine, canine, avian, feline and rabbit origins have been identified (for a review, see refs. 3,4,5,6). Baculovirus can transduce human embryonic stem cells⁷, mouse induced pluripotent stem (iPS) cells⁸ and mammalian cells cultured in 3D matrix⁹ and in suspension¹⁰. Within the baculovirus-transduced cells, transgenes can be expressed as long as they are driven by an appropriate promoter (e.g., cytomegalovirus (CMV) immediate-early or hybrid CAG promoter). Therefore, baculovirus has captured growing attention as a gene delivery vector for a plethora of applications, ranging from cell-based assay development¹¹, protein production¹², virus production¹⁰, virus-like particle production⁹, eukaryotic protein display¹³, RNA interference¹⁴, vaccine development^15,16 and cancer therapy¹⁷ to tissue engineering of bone^18,19, cartilage^20,21 and heart²².

Comparison of baculovirus with other viral gene delivery systems

In comparison with other viral gene delivery vectors such as retrovirus, lentivirus, adenovirus and adeno-associated virus (AAV), baculovirus has a number of desirable features:

• The natural host of baculovirus is insect cells, and thus baculovirus is nonpathogenic to humans and baculovirus neither replicates nor is toxic inside transduced mammalian cells²³.

• Baculoviral DNA degrades in the cells over time^24,25, and baculoviral DNA integration into host chromosomes is extremely rare and cannot be found^26,27 unless selective pressure is applied²⁸.

• Humans do not possess pre-existing antibodies²⁹ and T cells³⁰ specifically against baculovirus, which enables baculovirus to circumvent the pre-existing immunity problem.

• Recombinant baculovirus can be readily constructed and propagated to high titers by infecting its natural host insect cells in biosafety level 1 (BSL-1) facilities⁴.

• The baculovirus genome is large (∼134 kb) and hence it confers a huge cloning capacity of at least 38 kb (ref. 31).

The first three features ease safety concerns regarding the use of baculovirus for gene delivery, whereas the last two features simplify baculovirus vector production and expand the potential applications of baculovirus vectors.

However, there are three possible drawbacks associated with baculovirus vectors:

• Owing to the nonreplication and nonintegration nature, baculovirus typically mediates transient (<7 d (refs. 3,4,5,6)) expression, which may hamper the applications of baculovirus vectors in some scenarios requiring long-term, stable expression (e.g., hemophilia and X-linked severe combined immunodeficiency).

• Baculovirus is an enveloped virus and the baculoviral enveloped protein gp64 is essential for infection of insect cells and transduction of mammalian cells³². Consequently, baculovirus is vulnerable to mechanical forces, and bioprocessing steps during the vector purification (e.g., ultracentrifugation and filtration) tend to attenuate the virus titer.

• Baculovirus is inactivated by serum complement system³³, which may hamper the baculovirus administration by direct injection.

The advantages and disadvantages of baculovirus compared with other viral gene delivery vectors are further summarized in Table 1.

Table 1 Pros and cons of baculovirus and other viral vectors.

Key differences between our baculovirus transduction protocol and other protocols

Incubation with unconcentrated virus. For baculovirus transduction of permissive cells, frequently baculovirus supernatant is concentrated by ultracentrifugation and resuspended in PBS. Cells are then incubated with the virus for 1 h at 37 °C, using growth medium (e.g., DMEM) as the surrounding solution to adjust the final liquid volume^{2,34,35,36,37}. However, ultracentrifugation may result in considerable loss in virus titer. Moreover, larger transduction processes require larger amounts of baculovirus, yet virus preparation by ultracentrifugation constitutes a formidable task upon process scale-up.

To simplify the transduction process, in our protocol cells are incubated with unconcentrated virus (i.e., virus supernatant harvested from the infected insect cell culture) at a lower temperature (e.g., 25 °C or 27 °C) for 4–8 h with mild shaking, using PBS as the surrounding solution^24,38. This protocol enables and improves baculovirus-mediated gene transfer into HeLa^38,39, human BMSCs⁴⁰, BMSC-derived adipogenic progenitor cells⁴¹ and primary chondrocytes derived from rats²⁴ and rabbits^21,26, with efficiencies comparable or superior to those using concentrated virus. For instance, the transduction efficiencies of human BMSCs derived from umbilical cord blood can be elevated from ∼42% (using concentrated virus) to ∼73% (using our protocol)⁴⁰. Our protocol also ameliorates AcMNPV-mediated transduction of BHK⁹, Vero³⁹, canine MDCK⁴² and chicken Df-1 cells⁴², as well as augments the transduction of HEK293 and Schwann cells mediated by another baculovirus, Bombyx mori NPV⁴³. Because our protocol obviates the need for virus ultracentrifugation, it represents a simpler approach and also considerably reduces possible virus inactivation during ultracentrifugation.

Surrounding solution during transduction. Baculovirus titer often falls in the range of 3 × 10⁸ to 1 × 10⁹ plaque-forming units per milliliter (pfu/ml). Transduction of cells cultured in six-well plates (e.g., 3 × 10⁵ cells per ml) at a multiplicity of infection (MOI) of 100 (pfu per cell) only requires 30–100 μl of virus supernatant, and thus the virus supernatant needs to be diluted. For transduction in six-well plates, at least 500 μl of solution is required during the incubation period. Thus, virus supernatant can be diluted with insect cell medium to 100 μl and then mixed with the surrounding solution (400 μl) to a final volume of 500 μl. The volumetric ratio of diluted virus and surrounding solution can vary from 1:3 to 1:10, but in general a ratio of 1:4 is preferred (L.-Y.S., unpublished data).

The choice of surrounding solution has a considerable effect on transduction efficiency. We found that PBS as the surrounding solution confers better transduction efficiency and transgene expression than TNM-FH (the medium for baculovirus production) or DMEM^38,40. Comparison between the major components in PBS and these media reveals that NaHCO₃ present in DMEM and TNM-FH markedly inhibits baculovirus transduction by reducing the intracellular virus number⁴⁴.

NaHCO₃ is a buffering agent that is commonly added to the medium during medium preparation. Owing to the key role of NaHCO₃ and concerns that incubation of certain cells (e.g., stem cells) in PBS for hours may damage the cells, we modified the protocol by using NaHCO₃-deficient medium in lieu of PBS as the surrounding solution. By using our protocol, baculovirus is able to transduce various cell lines (e.g., Huh-7, HeLa and BHK), BMSCs²⁷, ASCs from humans (hASCs)⁴⁵ and rabbits (rASCs)⁴⁶ and even cell sheets derived from rASCs²², at efficiencies exceeding 95%.

Temperature and length of transduction. Temperature and the length of time that cells are incubated with baculovirus also dictate the transduction efficiency. Recombinant baculovirus is produced by infecting insect cells that are cultured using insect cell medium (e.g., TNM-FH) at 27 °C. We found that incubation of baculovirus and mammalian cells at 25 °C or 27 °C results in better transduction than at 4 °C or 37 °C, because the physical integrity of enveloped baculovirus is compromised at 37 °C, whereas virus particle adsorption to cell surface is less efficient at 4 °C (ref. 38). Incubation of cells with a virus supernatant for a prolonged period of time can enhance the transduction efficiency and transgene expression, as longer incubation time enables better virus entry³⁸. However, excessive incubation time at 27 °C may undermine cell health, and the optimal incubation time varies for different cells. In general, incubation for 4–6 h is recommended for most cell types²⁴.

Other parameters influencing baculovirus transduction efficiency

Reagents enhancing transgene expression. Baculovirus-mediated transgene expression can be elevated by the addition of histone deacetylase (HDAC) inhibitors such as sodium butyrate³⁷. HDAC inhibitors relax the chromatin structure by inducing histone hyperacetylation, which underscores the importance of epigenetic status of baculovirus genome for transgene expression. Notably, these drugs exert cytotoxicity at high concentrations⁴⁷, and the extent to which gene expression is upregulated hinges on cell lines⁴⁸. Therefore, optimal sodium butyrate concentration needs to be pre-determined for different cells.

Choice of promoter. Transgene expression is also promoter-dependent. Viral promoters that are active in mammalian cells, such as SV40, CMV, RSV and the hybrid CAG promoter, have been used to drive baculovirus-mediated gene expression with different strengths (for a review, see ref. 4). In addition, baculovirus-mediated transgene expression can be augmented by appending woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) to the 3′ end of transgene⁴⁹. WPRE is a cis-acting RNA element that can stabilize mRNA and facilitate mRNA nuclear processing and export of mRNA into the cytoplasm, and thus it enhances transgene expression. A three- to tenfold increase in baculovirus-mediated enhanced green fluorescent protein (EGFP) expression is noted in several cell lines⁴⁹.

Hybrid baculovirus vectors for prolonged transgene expression.

As mentioned above, baculovirus mediates transient transgene expression (typically <7 d), which may preclude the applications of baculovirus in certain scenarios requiring long-term expression. To prolong the expression, attempts to incorporate AAV inverted terminal repeats^7,34 or the Sleeping Beauty transposon^14,50 into baculovirus vectors have been made. These elements result in transgene integration into the host chromosome, and they can be used for sustained expression in neuronal cells and cancer cells.

In addition to developing integrating vectors, we have developed baculovirus systems that enable episomal maintenance of transgenes. Development of these systems is based on the hypothesis that shedding transgene cargo from the baculovirus genome to form an extrachromosomal DNA minicircle may prolong the transgene expression. With this assumption in mind, we established an FLP/Frt-based hybrid baculovirus system that comprises one baculovirus-expressing FLP recombinase and one substrate baculovirus harboring the transgene cassette flanked by two Frt sequences²⁷. Co-transduction with the two baculovirus vectors successfully extended the transgene expression in a number of mammalian cells, including rBMSCs²⁷ and rASCs⁵¹. The expression level and duration positively correlate with the recombination efficiency, presumably because minicircles confer stronger and longer transgene expression than their plasmid counterparts⁵². However, the FLP/Frt-mediated DNA minicircle formation occurs in only ∼40–50% of rBMSCs and rASCs^51,53. To further enhance the recombination efficiency, we have developed a new FLPo/Frt system⁴⁵ that explores the codon-optimized FLP (FLPo). The FLPo/Frt system enables recombination and minicircle formation in different mammalian cells, rASCs and rBMSCs at efficiencies >90%, and it has been successfully used in rASCs to extend and promote the expression of growth factors such as bone morphogenetic protein 2 (BMP-2)⁵⁴, BMP-6 (ref. 20), transforming growth factor β3 (ref. 20) and vascular endothelial growth factor (VEGF)²².

The new FLPo/Frt-based baculovirus system similarly consists of two baculoviruses: Bac-FLPo-expressing FLPo and the substrate baculovirus harboring the Frt-flanking transgene cassette (Fig. 1a). After co-transduction of cells, the expressed FLPo recognizes the Frt sites and excises the Frt-flanking cassette off the baculovirus genome, and hence it catalyzes the recombination and formation of episomal DNA minicircles encompassing the transgene cassette (Fig. 1b). The minicircle can persist in the cells for a longer term, whereas the baculovirus genome is rapidly degraded⁴⁵. Aside from FLPo, two other site-specific recombinases, Cre and codon-optimized ΦC31 (ΦC31o), have been explored⁴⁵. Cre catalyzes excision/recombination events between two identical loxP sites⁵⁵, whereas ΦC31o mediates excision/recombination between heterotypic sites attP and attB. Similarly to our work with the FLPo/Frt-based baculovirus system, we constructed a binary baculovirus vector system based on Cre/loxP (Fig. 1a) and ΦC31o/attP/attB (not shown). Upon co-transduction, the transgene cassette in the substrate baculovirus is excised by the recombinase (ΦC31o or Cre) expressed by a second baculovirus vector, and it recombines into minicircles⁴⁵.

Figure 1: Recombinase-based hybrid baculovirus system.

(a) Illustration of the FLPo/Frt- and Cre/loxP sytems. (b) Illustration of the FLPo-mediated recombination and DNA minicircle formation. After co-transduction with Bac-FLPo and the substrate baculovirus, the expressed FLPo recognizes the Frt sites flanking the transgene cassette, leading to the excision of the transgene cassette, recombination and the formation of the DNA minicircle. CMV, cytomegalovirus immediate early promoter; Wp, WPRE element; pA, polyadenylation signal.

Experimental design

Gene delivery into mammalian cells using baculovirus requires multiple stages: (i) construction of a transgene-containing donor plasmid, (ii) generation of recombinant bacmid, (iii) generation of recombinant baculovirus, (iv) virus amplification and (v) transduction. The construction of the donor plasmid is discussed here, but it is omitted from the procedure.

Construction of the donor plasmid. Baculovirus naturally infects insect cells, and recombinant baculoviruses are traditionally constructed by homologous recombination in two steps. A transgene is first cloned into a transfer plasmid under the control of a native baculovirus promoter (e.g., polh or p10). The second step involves co-transfection of the plasmid and linearized parental baculovirus DNA into insect cells, in which the overlapping sequences on the transfer plasmid and parental DNA lead to homologous recombination and hence transgene insertion into the baculoviral genome. Subsequent viral replication generates the recombinant baculovirus containing an integrated copy of the transgene. This baculovirus construction system is commercially available (e.g., BacPAK, Clontech).

To generate baculovirus vectors for transduction, we alternatively use the Bac-To-Bac system (Invitrogen), which exploits site-specific transposition of transgene cassette on the donor plasmid into the baculovirus shuttle vector (bacmid) in E. coli. The system offers a series of donor plasmids such as pFastBac 1 and pFastBac DUAL. The plasmids also contain the baculovirus promoter (e.g., polh or p10) upstream of the multiple cloning site and flanking attachment sites Tn7R and Tn7L. For expression in mammalian cells, the baculovirus promoter is simply replaced by a promoter (e.g., CMV) that is active in mammalian cells using standard cloning techniques⁵⁶ (designated as pBac-CMV5; Fig. 2), followed by cloning of the transgene into the donor plasmid.

Figure 2: Construction of recombinant baculovirus by site-specific transposition.

The baculovirus promoter in the pFastBac vector is replaced by the CMV promoter to yield pBac-CMV5, and the transgene is cloned into the plasmid. The resultant donor plasmid is transformed into E. coli DH10Bac, in which the transposase expressed by the helper plasmid transposes the Tn7-flanking cassette from the donor plasmid to the Tn7 attachment site on the bacmid, resulting in transgene integration into the bacmid and disruption of the lacZα gene. In the agar plate containing antibiotics, IPTG and X-gal, the recombinant bacmid can be isolated from the white colonies. The recombinant bacmid is transfected into insect cells, in which the recombinant bacmid results in recombinant virus generation.

To generate the FLPo/Frt-based hybrid baculovirus system, construction of two baculoviruses is required: Bac-FLPo and the substrate baculovirus harboring the Frt-flanking transgene cassette (Fig. 1a). Construction of the FLPo-expressing vector (Bac-FLPo) is straightforward, simply by cloning the optimized flpo gene from pPGKFLPobpA into the donor plasmid (pBac-CMV5) under the control of the CMV promoter. To construct the substrate vector harboring a transgene, a DNA fragment composed of a multiple cloning site flanked by two Frt sites is PCR-amplified from pLOI2226 (ref. 57). The amplicon (0.25 kb) is subcloned into pFastBac DUAL to yield pBac-Frt. Second, a CMV promoter (0.6 kb) is PCR-amplified from pcDNA3.1(+) and subcloned into pBac-Frt to yield pBac-Frt-CMV. Third, a WPRE sequence and an SV40pA are cloned into pBac-Frt-CMV to generate pBac-FCW. Fourth, the transgene is subcloned into pBac-FCW downstream of the CMV promoter.

To assay the recombination efficiency mediated by either ΦC31o, Cre or FLPo, we also generated a substrate baculovirus Bac-ALF, which harbors d2egfp (encoding destabilized EGFP, d2EGFP) flanked by tandem recombination sites (attP-loxP-Frt and attB-loxP-Frt)⁴⁵. To construct the donor plasmid pBacALF, the DNA fragment encoding the tandem recombination sites (XhoI-attP-loxP-Frt-BamHI-StuI-attB-loxP-Frt-HindIII) is first chemically synthesized and subcloned into pFastBac Dual to yield pALF. Second, the d2egfp-SV40pA fragment is PCR-amplified from pd2EGFP-N1 and inserted into the BamHI site of pALF to form pALF-dE. Finally, the CMV promoter (flanked by StuI/SmaI) is PCR-amplified from pcDNA3.1(+) and inserted into the StuI site of pALF-dE to yield pBacALF.

Generation of recombinant bacmid (Steps 1–33). To generate a recombinant bacmid, a donor plasmid is transformed into E. coli DH10Bac, which contains the bacmid and helper plasmid. The bacmid possesses a low-copy-number mini-F replicon (ori), a kanamycin resistance gene and a Tn7 attachment (att) site in frame within the lacZα gene. The helper plasmid within DH10Bac confers resistance to tetracycline and expresses the transposase (Fig. 2). After transformation of the recombinant donor plasmid into DH10Bac, the transposase transposes the Tn7-flanking cassette from the donor plasmid to the Tn7 attachment site, resulting in transgene integration into the bacmid and disruption of lacZα gene. The antibiotic selection and disruption of lacZα gene after transposition enable rapid selection/isolation of the recombinant bacmid from DH10Bac cultures. The recombinant bacmid is PCR-confirmed for the presence of transgene.

Production of recombinant baculovirus (Steps 33–44). The recombinant bacmid is used to transfect insect cells (e.g., Sf-9 derived from Spodoptera frugiperda), which leads to virus replication. Viral stocks collected from the transfected cells are defined as passage 0 (P0).

Amplification of recombinant baculovirus (Steps 45–56). The P0 baculovirus stocks can be used to further infect fresh Sf-9 cells for P1 virus production (Fig. 2). P1 virus can be used as an inoculum to infect Sf-9 cells for the generation of P2 virus stock. The resultant baculovirus is titrated by end-point dilution method⁵⁸ (Box 1). To evaluate transduction efficiency, baculovirus vectors expressing EGFP⁴⁷ and dsRed⁸ have been generated.

Transduction of mammalian cells (Step 57). In the PROCEDURE, we describe how to transduce cell lines (Step 57A, describing transduction of HEK293 as an example), iPS cells (Step 57B), rASCs (Step 57C) and a rASC sheet (Step 57D). The procedures for HEK293 and iPS cell culture are routine, and they have been described previously (see refs. 59 and 60 for iPS cells); thus, this protocol starts from the cell seeding. rASCs can be harvested s.c. from the inguinal fat pads surrounding the epididymis of 3–4-month-old New Zealand white (NZW) rabbits (see Box 2 for a detailed protocol)⁵¹.

For transduction, cells are seeded on to culture plates or flasks (e.g., six-well plates, 10-cm dishes, T-75 or T-150 flasks) and incubated overnight. At day 2, a certain volume (depending on the multiplicity of infection (MOI) and baculovirus titer) of unconcentrated baculovirus supernatant is diluted with fresh insect cell (Sf-9) culture medium, and it is then mixed with NaHCO₃-free culture medium (e.g., DMEM or α-MEM) at a volumetric ratio of 1:4. The final volume of virus solution should be sufficient to wet the surface. Larger volumes of virus solution may reduce the chance of virus contact with cells and may decrease the transduction efficiency. Transduction is initiated by directly adding the appropriate volume (e.g., 500 μl per well in six-well plates, 4 ml per 10-cm dish and 5 ml per T-75 flask) of virus solution to cells. The transduction continues by gentle shaking on a rocking plate at room temperature for 4–6 h. After the transduction period, virus solution is replaced by the culture medium containing sodium butyrate. After ∼15–24 h of incubation at 37 °C, the medium is replaced by fresh medium. At day 3 (1 d post transduction, 1 dpt), cells are collected for analysis or applications (Fig. 3). For instance, cells transduced with Bac-CE (expressing EGFP) can be harvested at 1 dpt and analyzed by flow cytometry for the percentage of GFP⁺ cells (i.e., transduction efficiency). Alternatively, the cells continue to be cultured with medium exchange every 2–3 d (Fig. 3). For mock-transduction control, the procedure is the same, except that insect cell culture medium is used in lieu of virus supernatant.

Figure 3

Transduction of cells in a six-well plate.

Box 1: Titration of recombinant baculovirus • TIMING 12–15 d

Box 2: Isolation and culture of rASCs • TIMING 21–23 d

Limitations of the protocol

The Bac-to-Bac system we commonly use eliminates the need for multiple rounds of plaque purification, which reduces the time required to obtain the P0 recombinant virus. However, the transgene in the genome of bacmid-derived baculovirus is more prone to spontaneous excision, leading to a rapid decrease in transgene expression in passages beyond P8 (ref. 61). Therefore, it is advised to keep the virus passage low.

Baculovirus transduction is highly efficient for a wide variety of cell types, yet it is reported that baculovirus transduction of hematopoietic cell lines such as U937, K562, Raw264.7, LCL-cm and Raji is inefficient (for a review, see ref. 6). In addition, baculovirus transduction efficiency for primitive stem cells such as embryonic stem cells⁶² and iPS cells⁸ is relatively low (<40–50%). Furthermore, baculovirus-mediated growth factor expression is relatively inefficient in rat or mouse ASCs or BMSCs (K.-C.L. and C.-L.Y., unpublished data), although the expression is robust in rASCs or rBMSCs^53,54.

Last, although the transgene expression can be prolonged from <7 d to ∼3 weeks using the FLPo/Frt-based hybrid baculovirus vectors, for longer transgene expression (months to years) integration of transgene cassette is required. In this regard, hybrid baculovirus vectors by incorporating AAV inverted-terminal repeats^7,34 or Sleeping Beauty transposons^14,50 have been developed to enable transgene integration into the chromosome and long-term expression.

Materials

REAGENTS

• MAX Efficiency DH10Bac chemically competent cells (Invitrogen, cat. no. 10361-012)

• Sf-9 cells (ATCC, cat. no. CRL-1711)

• Mammalian cells to be transduced, for example, HEK293 cells (ATCC, cat. no. CRL-1573), iPS cells (we have used the mouse iPS cell line 20D17, supplied by S. Yamanaka, Center for iPS Cell Research and Application, Kyoto University) or rASCs (derived as described in Box 2). See INTRODUCTION for a full discussion of the cell types that we have successfully transduced

• S.O.C. medium (Invitrogen, cat. no. 15544-034)

• LB broth (BD Biosciences, cat. no. 244620)

• Agar (bacteriological; OXOID, cat. no. LP0011)

• Sf-900 II serum-free medium (SFM; Invitrogen, cat. no. 10902-088)

• TNM-FH insect cell medium (Sigma, cat. no. T3285)

• DMEM (high glucose; Sigma, cat. no. D5648)

• DMEM (low glucose; Gibco, cat. no. 31600-034)

• α-MEM (Gibco, cat. no. 12000-022)

• PBS without calcium or magnesium (pH 7.4; Invitrogen, cat. no. 14190-094)

• Hyclone FBS (Thermo Scientific, cat. no. SH30070.03)

• Sodium bicarbonate (NaHCO₃; Sigma, cat. no. S5761)

• Gentamycin (AMRESCO, cat. no. 0304)

• Kanamycin (AMRESCO, cat. no. 0408)

• Tetracycline (AMRESCO, cat. no. 0422)

• X-gal (AMRESCO, cat. no. 0428)

• IPTG (AMRESCO, cat. no. 0487)

• Absolute 2-propanol (Sigma, cat. no. I9516)

• Cellfectin II reagent (Invitrogen, cat. no. 10362-100)

• Trypsin-EDTA (0.05% (wt/vol); Gibco, cat. no. 25300)

• Tris-HCl (Sigma, cat. no. T5941)

• EDTA (Sigma, cat. no. E6758)

• Sodium hydroxide (NaOH; Sigma, cat. no. S8045)

• SDS (Sigma, cat. no. L3771)

• Potassium acetate (Sigma, cat. no. P1190)

• DMSO (Sigma, cat. no. D2650)

• Trypan blue stain (0.4%; APOLO, cat. no. APL-0167)

• Ethanol (70% (vol/vol); Sigma, cat. no. 02877)

• Gelatin (0.1% (wt/vol) solution) from porcine skin type A (Sigma, cat. no. G1890)

• MEM non-essential amino acids solution, 100× (Gibco, cat. no. 11140-050)

• 2-mercaptoethanol (55 mM; Gibco, cat. no. 21985-023)

• Leukemia inhibitory factor (10⁷ U/ml; LIF, Millipore, cat. no. ESG1107)

• Type I collagenase (Gibco, cat. no. 17100-017)

• RNase A (Sigma, cat. no. R6513)

• Penicillin-streptomycin, 100× (10,000 U/ml; Gibco, cat. no. 15140-122)

• Penicillin-streptomycin-amphotericin B solution (Biological Industries, cat. no. 03-033-1B)

• Sodium butyrate (Sigma, cat. no. B5887)

• pFastBac vectors such as pFastBac 1 (Invitrogen, cat. no. 10360-014) and pFastBac Dual (Invitrogen, cat. no. 10712-024)

• Recombinant baculovirus expressing EGFP (Bac-CE), DsRed (Bac-ER), FLPo (Bac-FLPo) and BMP-2 (Bac-FCBW) under the control of the CMV promoter

EQUIPMENT

• Incubator, set at 37 °C, 5% CO₂ (Thermo Scientific)

• Incubator, 27 °C, no CO₂ (Firstek Scientific, RI-102)

• Shaking incubator, 27 °C, no CO₂ (Cocono)

• Upright ultra-low-temperature freezers, set at −80 °C (Forma 900 series, Thermo Scientific)

• Refrigerator, 4 °C (Daytime)

• Freezer, −20 °C (Elcold)

• Water bath (Firstek Scientific)

• Rotator (TAITEC, RT-50)

• Laminar flow cabinet (UBI)

• Sterilizer (Tomin, High-Pressure Steam Sterilizer)

• Inverted microscope equipped with a phase-contrast ring and fluorescence filters (Nikon, TE200)

• Upright microscope (Nikon)

• Microtube (1.5 ml; Axygen, MCT-150-C)

• Tubes (15 and 50 ml; Corning, cat. nos. 43079 and 35276, respectively)

• Disposable culture tube (Kimble Chase, cat. no. 73500-16100)

• Petri dish (Protech, cat. no. PT-PD-90)

• Tissue culture plates (6-, 12- and 96-well; Thermo Scientific, cat. nos. 140675, 167008 and 150628, respectively)

• Tissue culture flasks (T-75 and T-150; Thermo Scientific, cat. nos. 156499 and 159910, respectively)

• Tissue culture centrifuge (Hettich Universal 32)

• Microcentrifuge (Hettich Zentrifugen MIKRO-120)

• Flow cytometer (BD Biosciences, FACSCalibur)

• Rocking plate (TKS, RS01)

• Vortex mixer (Scientific Industries, cat. no. SI-0236)

• Multi-place magnetic stirrer (Thermolyne, Cellgro 45700)

• Spinner flask (250 ml; Bellco, cat. no. 1965-80255)

• Hemocytometer (Marienfeld-Superior, cat. no. 6300-30)

• Pipettes (5 and 10 ml; Costar, cat. nos. 4487 and 4488, respectively)

• Tips (10, 200 and 1,000 μl; MultiMax, cat. nos. 7230, 2947 and 2950, respectively)

• Pipettors (P2N, P10N, P20N, P200N and P1000N; Gilson, cat. nos. F144561, F144562, F144563, F144565 and F144566, respectively)

• Pipette aid

• Surgical scissors

• Surgical forceps

• Shaver

• Scalpel

• Induction chamber

• Glass cell spreaders

• Laboratory glass bottle (250 ml; Schott Duran, cat. no. 2180136)

• Cryogenic vials (Nalgene, cat. no. 5000-0020)

• Cryo box

• Syringes (BD Biosciences, 1-ml syringe with a 26-G needle for anesthesia)

• Bottle-top filters (0.22-μm; Sartorius, cat. no. 9.049 205)

• Syringe filter (0.22-μm; Nalgene, cat. no. 190-2520)

REAGENT SETUP

LB broth

• To prepare 1,000 ml of LB broth, dissolve 25 g of the LB broth powder in 1,000 ml of distilled water, and mix it thoroughly. Autoclave the medium at 121 °C for 15 min. Cool the broth to room temperature, and then store it at 4 °C for up to 3 months.

LB agar plate

• Dissolve 25 g of LB broth powder and 15 g of agar powder in 1,000 ml of distilled water and mix it thoroughly. Autoclave the solution at 121 °C for 15 min. When the temperature of the heated solution drops to 55 °C, add gentamycin, kanamycin, tetracycline, X-gal and IPTG to final concentrations of 7, 50, 10, 200 and 40 μg/ml, respectively. Divide the agar mixture into separate Petri dishes (20 ml per dish) and cool the plates to room temperature. Store the plates at 4 °C for up to 1 month.

Resuspension buffer

• To prepare 100 ml of resuspension buffer, dissolve 0.24 g of Tris-HCl and 0.29 g of EDTA in 80 ml of distilled water, and then adjust the pH to 8.0. Add 10 mg of RNase A and fill the container with distilled water to a final volume of 100 ml. Store the buffer at 4 °C and use it within 1 month.

Lysis buffer

• To prepare 100 ml of lysis buffer, dissolve 0.8 g of NaOH and 1 g of SDS in 100 ml of distilled water. Store the buffer at room temperature and use it within 3 months. Mix the buffer thoroughly before use.

  Critical

  If precipitates form in the lysis buffer, warm the buffer in a 37 °C water bath until the precipitates totally dissolve.

Precipitation buffer

• To prepare 100 ml of precipitation buffer, dissolve 29.4 g of potassium acetate in 50 ml of distilled water and adjust the pH to 5.5. Fill with distilled water to a final volume of 100 ml. Store the buffer at room temperature and use it within 3 months.

Elution buffer

• To prepare 100 ml of elution buffer, dissolve 0.16 g of Tris-HCl and 0.03 g of EDTA in 80 ml of distilled water, adjust the pH to 8.0, and use distilled water to bring the final volume to 100 ml. Store the buffer at room temperature and use it within 1 month.

Sf-9 medium

• To prepare 1,000 ml of Sf-9 medium, mix 900 ml of TNM-FH medium (filtered through 0.22-μm) with 100 ml of FBS. Store the medium at 4 °C for up to 1 month.

HEK293 medium

• To prepare 500 ml of HEK293 medium, mix 450 ml of DMEM (high glucose) medium (filtered through a 0.22-μm filter) and 50 ml of FBS. Store the medium at 4 °C for up to 1 month.

Gelatin-coated culture flask and plate

• Add 0.1% (wt/vol) gelatin solution to a T-75 flask (5 ml) or a six-well plate (1 ml). Ensure that the entire bottom surface is covered with liquid. Incubate the flask or plate at 37 °C for 30 min. Aspirate the gelatin solution before use. Prepare the flask or plate immediately before use.

iPS medium

• To prepare 500 ml of iPS medium, mix 419 ml of DMEM (high glucose) medium (filtered through a 0.22-μm filter), 75 ml of FBS (15% (vol/vol), 5 ml of 100× non-essential amino acids, 0.9 ml of 2-mercaptoethanol and 50 μl of LIF. Store the medium at 4 °C and use it within 1 month.

rASC medium

• rASC medium can be DMEM- or α-MEM–based. To prepare 500 ml of rASC medium, mix 445 ml of DMEM (low glucose) medium or α-MEM medium (filtered through a 0.22-μm filter), 50 ml of FBS (10% (vol/vol) and 5 ml of 100× penicillin-streptomycin. Store the medium at 4 °C for up to 1 month.

rASC sheet fabrication medium

• To prepare 500 ml of rASC sheet fabrication medium, mix 395 ml of α-MEM medium (filtered through a 0.22-μm filter), 100 ml of FBS (20% (vol/vol)) and 5 ml of 100× penicillin-streptomycin. Store the medium at 4 °C for up to 1 month.

Surrounding solution

• Surrounding solution is culture medium containing 10% (vol/vol) FBS, without NaHCO₃. The surrounding medium should be stored at 4 °C and used within 1 month.

  Critical

  The surrounding medium is prepared in a similar manner to the culture medium, except that 3.7 grams per liter NaHCO₃ is not added.

Type I collagenase solution

• To prepare 5 ml of 2% (wt/vol) type I collagenase, dissolve 0.1 g of type I collagenase powder in a final volume of 5 ml of PBS solution. Type I collagenase solution should be prepared freshly before starting the isolation process, and it should be filter-sterilized using a 0.22-μm syringe filter.

Sodium butyrate stock solution

• To prepare 100× stock solution of sodium butyrate (300 mM), dissolve 1 g of sodium butyrate in 30.3 ml of distilled water and sterilize the solution by passing it through a 0.22-μm filter. Freeze the stock solution in aliquots at −20 °C and use it within 6 months.

Procedure

Generation of recombinant bacmid

Timing 5 d

Critical Step

If a recombinant baculovirus stock is already available, Steps 1–44 can be skipped.

• 1 Transposition of pFastBac donor plasmid, day 1. Dilute the donor plasmid to 10 ng/μl using sterile water in a sterile 1.5-ml microtube.

• 2 Thaw one vial of MAX Efficiency DH10Bac competent cells on ice.

• 3 Prechill a sterile 1.5-ml microtube on ice.

• 4 Add 30 μl of DH10Bac competent cells into the prechilled microtube and keep it on ice.

• 5 Add 1 μl of the diluted donor plasmid into 30 μl of DH10Bac competent cells.

• 6 Incubate the competent cells on ice for 20 min.

  Critical Step

  Do not hold the bottom of the microtube with your finger. Hold the upper part of the microtube to avoid warming the competent cells.

• 7 Incubate the mixture in the 42 °C water bath for 60 s to perform heat shock.

• 8 Immediately transfer the mixture to ice and leave it on ice for 5 min.

• 9 Add 500 μl of S.O.C. medium that has been prewarmed to room temperature into the microtube containing competent cells. Shake the tube in a rotator (250 r.p.m.) at 37 °C for 4 h. Thirty minutes before the end of this 4-h incubation, prewarm an LB agar plate at 37 °C for 30 min.

• 10 Centrifuge the cells at 9,000g for 2 min at room temperature. Remove 470 μl of supernatant (leaving 60 μl). Resuspend the cell pellet by pipetting gently.

• 11 Spread 60 μl of the resuspended competent cells on the prewarmed LB agar plate using a glass cell spreader.

• 12 Incubate the LB agar plate at 37 °C for 48 h for colony formation. Store the plate at 4 °C before the next step if necessary.

  Critical Step

  Normally, blue and white colonies are formed after incubation. Successful transgene cassette transposition into the attachment site disrupts the lacZα gene, leading to interrupted expression of LacZα peptide and the formation of white colonies. Without transposition, lacZα gene remains intact, giving rise to LacZα peptide expression and the formation of blue colonies in the presence of X-gal in the LB agar plate. Therefore, colonies containing the recombinant bacmid are white in a background of blue colonies that harbor the unaltered bacmid.

  Critical Step

  The process may be accelerated by incubating the agar plate for only 16–24 h. However, at this time, colonies are relatively small. For the following bacmid isolation, pick the largest, most isolated white colonies to avoid selecting false positives. Avoid picking colonies that appear gray or are darker in the center, as they can contain a mixture of cells with empty bacmid and recombinant bacmid.

  Pause point

  The LB agar plate can be stored at 4 °C for 1 month.

• 13 Day 3. Prewarm the spread LB agar plate to room temperature.

• 14 Prewarm another fresh LB agar plate at 37 °C for 30 min and divide the fresh LB agar plate into three areas with a marker.

• 15 Pick a blue colony (as control) and two white colonies onto the spread LB agar plate and re-streak them on the three areas of the fresh LB agar plate.

• 16 Incubate the re-streaked LB agar plate at 37 °C for 16–24 h for colony formation. Store the plate at 4 °C before the next step if necessary.

  Critical Step

  The white colonies should contain the recombinant bacmid. Verify the integration of the transgene cassette into bacmid by restriction analysis or PCR if necessary.

  Pause point

  The re-streaked LB agar plate can be stored at 4 °C for 1 month.

• 17 Isolation of recombinant bacmid DNA, day 4. Add 3 ml of LB broth containing gentamycin (7 μg/ml), kanamycin (50 μg/ml) and tetracycline (10 μg/ml) into a disposable culture tube.

• 18 Pick a white colony from the re-streaked LB agar plate, and then inoculate the cells into the disposable culture tube.

  Critical Step

  We recommend that you culture two white colonies individually in LB broth to ensure successful isolation of recombinant bacmids.

• 19 Place the culture tube on the rotator (250 r.p.m.) at 37 °C for 20 h.

• 20 Day 5. Transfer 1 ml of cultured bacterial cells to a 1.5-ml microtube and centrifuge the tube at 9,000g for 2 min at room temperature. Remove all of the supernatant.

• 21 Add 200 μl of resuspension buffer to the pellet. Resuspend the cells by pipetting or vortexing.

• 22 Add 200 μl of lysis buffer to the cell suspension. Mix it gently by inverting the microtube ten times.

• 23 Incubate the mixture at room temperature for 5 min.

• 24 Add 300 μl of precipitation buffer to the mixture. Mix it immediately by inverting the microtube ten times. Do not vortex.

• 25 Chill the microtube on ice for 10 min. Centrifuge the mixture at 15,000g at 4 °C for 20 min.

• 26 Transfer 600 μl of the supernatant to another 1.5-ml microtube.

  Critical Step

  Avoid pipetting any white precipitate in the supernatant. Pipette the clear supernatant into the microtube and centrifuge it at 15,000g for another 5 min at 4 °C to remove any contaminating precipitates.

• 27 Add 800 μl of absolute 2-propanol to the clear supernatant. Mix it gently by inverting the microtube ten times.

• 28 Chill the microtube on ice for 10 min. Centrifuge the tube at 15,000g at 4 °C for 15 min. Remove the supernatant.

• 29 Add 500 μl of 70% (vol/vol) ethanol to the pellet. Mix gently by inverting the microtube ten times.

• 30 Centrifuge the tube at 15,000g at 4 °C for 5 min. Remove the supernatant completely.

• 31 Open the cap of the microtube and air-dry the DNA pellet for 5–10 min until the pellet becomes half-transparent.

• 32 Resuspend the DNA pellet in 40 μl of elution buffer by gentle pipetting. Allow the pellet to dissolve completely on ice or in a 4 °C refrigerator.

  Critical Step

  To avoid shearing the bacmid DNA, do not pipette more than two times.

• 33 Divide the bacmid DNA into aliquots in separate sterile 1.5-ml microtubes, and store them at −20 °C to avoid more than one freeze-thaw cycle.

  Pause point

  The bacmid DNA can be stored at −20 °C for up to 2 weeks.

Production of recombinant baculovirus

Timing 5–7 d

• 34 Transfection of recombinant bacmid DNA, day 1. Grow Sf-9 cells with Sf-9 medium (containing 10% (vol/vol) FBS) in suspension to log-phase (e.g., 1–2 × 10⁶ cells per ml) with >95% viability.

  Critical Step

  Sf-9 cells can be readily cultured in monolayer or in suspension at 27 °C without CO₂.

• 35 Dilute Sf-9 cells to 3 × 10⁵ cells per ml and seed 1 ml of cells to each well of a 12-well plate. Incubate the cells in a 27 °C incubator for at least 30 min to allow cell attachment.

• 36 For each bacmid that is ready for transfection, prepare solutions A and B (containing bacmid) in a sterile 1.5-ml microtube as follows:

  Table 3

  Solution A		Solution B
  Reagent	Volume (μl)	Reagent	Volume (μl)
  Cellfectin II reagent	2	Bacmid DNA	5
  Sf-900 II SFM	30	Sf-900 II SFM	30

  Critical Step

  SFM is used in transfection because proteins in the FBS interfere with the Cellfectin reagent and inhibit the transfection.

• 37 Mix gently solutions A and B in a sterile 1.5-ml microtube, and incubate it at room temperature for 30 min.

• 38 Add 240 μl of Sf-900 II SFM to the DNA-lipid mixture and mix it thoroughly.

• 39 Wash the attached Sf-9 cells with Sf-900 II SFM twice to remove FBS. Discard the medium completely.

• 40 Add the DNA-lipid mixture to the 12-well plate (300 μl per well). Incubate the 12-well plate at 27 °C for 5 h for transfection.

  Critical Step

  Do not add antibiotics during transfection.

• 41 After transfection, discard the DNA-lipid mixture in the well. Add 1 ml of Sf-9 medium containing 10% (vol/vol) FBS and 1% (vol/vol) penicillin-streptomycin-amphotericin B to each well. Incubate the 12-well plate at 27 °C for 4–6 d.

• 42 Day 5–7. Visually observe the cytopathic effect (CPE) of transfected Sf-9 cells under a microscope. When CPE is observed in ∼30% of cells, collect the culture medium in a sterile 1.5-ml microtube.

  Critical Step

  At 3 d after transfection, visually check the CPE of transfected cells (e.g., cell lysis, for which cells appear lysed resulting in clearing plaques on the cell monolayer). If transfection efficiency is low, CPE may not be obvious until 4 or 5 d after transfection.

  Troubleshooting

• 43 Centrifuge the culture medium at 500g for 10 min at room temperature. Pipette the supernatant into a sterile 1.5-ml cryogenic vial as the P0 virus.

• 44 Divide the P0 virus into separate sterile 1.5-ml cryogenic vials. Store the working stock at 4 °C and the master stock at −80 °C for long-term storage.

  Critical Step

  Do not store the virus stock at −20 °C, which leads to more rapid titer loss⁶³. Protect the stock from light.

  Pause point

  The P0 virus can be stored for at least 3 months at 4°C and for years at −80 °C.

Amplification of recombinant baculovirus

Timing 10–12 d

• 45 Day 1. Grow Sf-9 cells using Sf-9 medium to log phase (∼1 × 10⁶ cells per ml). Seed 10 ml of Sf-9 cells to a T-75 flask. Incubate the cells at 27 °C for at least 30 min to allow cell attachment.

  Critical Step

  For baculovirus amplification, Sf-9 medium containing 10% (vol/vol) FBS is recommended because baculovirus is more stable in the presence of FBS.

• 46 Aspirate 5 ml of medium from the T-75 flask to a 15-ml centrifuge tube. Add 25 μl of P0 virus stock to the medium and mix it thoroughly.

• 47 Transfer the 5-ml medium containing P0 virus back to the T-75 flask. Continue the infection by incubating the flask in a 27 °C humidified incubator for 4–5 d.

  Critical Step

  Infecting cells at low MOI (e.g., 0.05–0.1) is recommended for baculovirus amplification. Infecting cells with higher MOI may generate noninfectious defective virus particles and it may reduce the quality of the baculovirus stock. At this stage, we do not routinely determine the virus titer, because it takes 12–15 d. Instead, we assume a virus titer of 2 × 10⁷ pfu/ml according to our experience, and add 25 μl of virus stock to the T-75 flask. Do not directly add the virus stock into the T-75 flask. We recommend premixing the virus stock with 5 ml of Sf-9 medium before infection.

• 48 Day 5 (−6). Check the CPE of infected Sf-9 cells. When CPE (e.g., cell lysis) is observed in ∼70% of cells, collect the culture supernatant in a sterile 15-ml centrifuge tube.

• 49 Centrifuge the cells at 500g for 10 min at room temperature. Pipette the supernatant into another sterile 15-ml centrifuge tube as the P1 virus stock.

• 50 Store the P1 virus stock at 4 °C and protect it from light. For long-term storage, transfer the virus stock into separate 1.5-ml cryogenic vials and store them at −80 °C.

  Pause point

  The P1 virus can be stored at −80 °C for years. When storing at 4 °C, the virus is stable for at least 3 months.

• 51 Day 6 (−7). To generate P2 virus stock, grow 200 ml of Sf-9 cells in the 250-ml spinner flask to log phase (∼1.0–1.2 × 10⁶ cells per ml) using Sf-9 medium.

• 52 Transfer 10 ml of culture from the spinner flask to a 15-ml centrifuge tube. Add 50 μl of the P1 virus stock to the 10-ml culture.

  Critical Step

  The titer (pfu/ml) of baculovirus can be determined using either plaque assay⁵⁸ or end-point dilution assay, which is described in detail in Box 1. If the virus titer is not determined at this stage, a virus inoculum volume of 50 μl (assuming a P1 virus titer of 2 × 10⁸ pfu/ml) is recommended. Premix the virus stock with 10 ml of culture.

• 53 Transfer the 10-ml culture containing P1 virus back to the spinner flask. Place the spinner flask on the magnetic stirrer (100 r.p.m.) in a 27 °C humidified incubator and incubate it for 4–5 d.

• 54 Days 10–12. Check the viability of infected Sf-9 cells by trypan blue dye exclusion with a hemocytometer. When the cell viability drops to ∼70%, collect the culture in four sterile 50-ml centrifuge tubes.

• 55 Centrifuge at 500g for 10 min at room temperature. Transfer the supernatant to a sterile 250-ml laboratory glass bottle as the P2 virus stock.

• 56 Store the P2 virus stock at 4 °C and protect it from light. Titrate the virus as described in Box 1.

  Critical Step

  For mammalian cell transduction, in general, a batch of 100–200 ml of P2 virus stock is sufficient for experiments.

  Troubleshooting

  Pause point

  The P2 virus can be stored at 4 °C for at least 3 months.

Transduction of mammalian cells with baculovirus

• 57 To transduce general cell lines (e.g., Huh-7, SNU-449, A549, HeLa and HEK293), follow option A. Option A describes the transduction of HEK293 cells in six-well plates using a baculovirus (Bac-CE) expressing EGFP as an example. To transduce iPS cells, follow option B, which describes the transduction of iPS cells using Bac-ER (containing a EF1α-DsRed transgene cassette) as an example. To transduce rASCs using the FLPo/Frt-based hybrid system, follow option C. To transduce rASC sheets, follow option D. Option D demonstrates the fabrication and transduction of rASC sheet using Bac-CE.

  • A Transduction of a general cell line

    • i Day 1. Trypsinize HEK293 cells from the T-75 flask and resuspend the cells with prewarmed HEK293 medium to a density of 1.5 × 10⁵ cells per ml.

      Critical Step

      We prefer not to add antibiotics, as they mask bacterial contamination. Nonetheless, antibiotics do not affect baculovirus transduction, and they can be used in primary cells such as rASCs.

    • ii Seed 2 ml of cell suspension into each well of a six-well plate (3 × 10⁵ cells per well). Incubate the cells overnight at 37 °C. After overnight incubation, HEK293 cells should reach 60–70% confluence.

      Critical Step

      The initial seeding cell may slightly vary from cell line to cell line, depending on the cell size.

    • iii Day 2. Calculate the required virus volume based on the virus titer (typically 3 × 10⁸–1 × 10⁹ pfu/ml for P2 virus) and cell number (3 × 10⁵ cells per well). For each well, the virus volume required is calculated using the following formula: volume required (ml) = desired MOI (pfu per cell) × cell number (cells)/virus titer (pfu/ml).

    • iv For each well, add the required volume of Bac-CE into Sf-9 medium to a total volume of 100 μl. Next, mix 100 μl of virus solution with 400 μl of surrounding solution (NaHCO₃-free HEK293 medium).

      Critical Step

      For example, when 3 × 10⁵ cells are transduced by Bac-CE with a titer of 5 × 10⁸ pfu/ml at MOI 100, the volume required for each virus supernatant is 100 (pfu per cell) × 3 × 10⁵ (cells)/5 × 10⁸ (pfu/ml) = 0.06 ml (60 μl). Add 60 μl of Bac-CE to 40 μl of Sf-9 medium. Mix the 100 μl of virus mixture with 400 μl of surrounding solution. For mock transduction as control, simply mix 100 μl of Sf-9 medium with 400 μl of surrounding solution.

      Critical Step

      If the calculated total virus volume exceeds 100 μl, increase the surrounding solution volume with the fixed volumetric ratio (virus solution:surrounding solution = 1:4).

      Critical Step

      If a larger vessel (e.g., a T-75 or T-150 flask) is used, the volume of the final virus solution is proportionally increased according to the culture area.

    • v Discard the HEK293 medium and wash the cells twice with PBS (1 ml per well).

    • vi Remove PBS and initiate the transduction by adding 500 μl of the mixed virus solution into each well.

    • vii Put the six-well plate on a rocking plate. Continue the transduction process by gently shaking the plate (10 times per min) for 6 h at room temperature.

      Critical Step

      Transduction efficiency depends on cell type, MOI and incubation time, which may vary from 4 to 6 h. For HEK293, transduction at MOI 100 for 6 h at room temperature yields transduction efficiency >95% without compromising cell viability. Incubation at 37 °C results in reduced transduction efficiency because baculovirus integrity is compromised at 37 °C.

    • viii Aspirate the virus solution and wash the cells again. Add fresh HEK293 medium containing 3 mM sodium butyrate. Incubate the mixture at 37 °C for 24 h.

      Critical Step

      Sodium butyrate enhances transgene expression, but high sodium butyrate concentration is toxic to cells. We determine that 3 mM enhances the transduction of HEK293 without compromising cell viability.

    • ix Day 3. Observe the fluorescence-emitting cells under the fluorescence microscope. Trypsinize and resuspend cells in 0.5 ml of PBS. Analyze the percentage of fluorescence-emitting cells using a flow cytometer.

      Troubleshooting

    Timing 3 d

  • B Transduction of iPS cells

    • i Day 1. Seed iPS cells to a gelatin-coated six-well plate (2 × 10⁵ cells per well, 2 ml). Keep the cells at 37 °C overnight.

    • ii Day 2. Calculate the required virus volume as described in Step 57A(iii).

    • iii Remove the medium and gently wash the cells twice with PBS (1 ml per well).

    • iv For each well, add the required volume of baculovirus (Bac-ER) into Sf-9 medium to a total volume of 100 μl. Next, mix the 100 μl of virus solution with 400 μl of surrounding solution (NaHCO₃-free iPS medium).

      Critical Step

      The iPS cells express GFP under the control of the Nanog promoter. Therefore, here we transduce the cells with a baculovirus expressing DsRed (emitting red fluorescence).

    • v Remove PBS from the six-well plate. Add the mixed virus solution to the wells (500 μl per well).

    • vi Gently shake the six-well plate on a rocking plate (10 times per min). Continue the transduction process for 4 h at room temperature.

      Critical Step

      iPS cells do not adhere firmly to the plate. Treat the cells gently.

    • vii After transduction, aspirate the baculovirus-containing solution and gently wash the cells twice with 1 ml of PBS. Replenish with 2 ml of iPS medium containing 3 mM sodium butyrate. Incubate the cells at 37 °C for 24 h.

    • viii Day 3. Observe the DsRed expression under a fluorescence microscope. Trypsinize and resuspend the cells in 0.5 ml of PBS. Analyze the percentage of fluorescence-emitting cells using a flow cytometer (543 nm).

      Troubleshooting

    Timing 3 d

  • C Transduction of rASCs

    • i Day 1. Move the cryogenic vial containing rASCs (1 × 10⁶ cells per vial) from liquid nitrogen. Thaw the cells in a 37 °C water bath. Be sure not to immerse the cap.

    • ii Once the crystals melt (1–2 min), transfer the cells to a T-75 flask containing 9 ml of prewarmed rASC medium. Keep the cells at 37 °C in a 5% CO₂ incubator.

    • iii After 6 h of attachment, replace the DMSO-containing medium with 10 ml of rASC medium. Culture the cells at 37 °C for 2–3 d until ∼80–90% confluency.

      Critical Step

      As rASCs tend to differentiate upon 100% confluency, do not allow the cells to grow to full confluency. We recommend subculturing rASCs at ∼80–90% confluency.

    • iv Day 3 (−4). Wash the cells with 10 ml of PBS twice, and discard the solution. Add 1 ml of prewarmed 0.05% (wt/vol) trypsin-EDTA to the T-75 flask and incubate it at 37 °C for 5 min to detach rASCs. Check for complete cell detachment by examining cell morphology under a microscope.

    • v Inactivate trypsin by adding 5 ml of rASC medium and gently resuspending the cells. Aspirate 50 μl of cell solution for counting (the concentration is generally 4–6 × 10⁵ cells per ml).

    • vi Dilute the cell suspension to 1 × 10⁵ cells per ml with rASC medium and seed 2 ml of cell solution to the six-well plate (2 × 10⁵ cells per well or ∼2 × 10⁴ cells per cm²). Incubate the cells at 37 °C overnight. After overnight incubation, the rASC density is ∼2 × 10⁵ cells per well (∼60–70% confluency).

      Critical Step

      For rASC transduction in a larger scale, seed rASCs to T-75 or T-150 flasks with the same cell density (∼2 × 10⁴ cells per cm²) and incubate them overnight in the same manner.

    • vii Day 4 (−5). Calculate the required virus volume as described in Step 57A(iii). For example, when 2 × 10⁵ cells are co-transduced by Bac-FLPo and the substrate baculovirus with the same titer (e.g., 5 × 10⁸ pfu/ml) at the same MOI (e.g., 50), the volume required for each virus supernatant is 50 (pfu per cell) × 2 × 10⁵ (cells)/5 × 10⁸ (pfu/ml) = 0.02 ml (20 μl). Add 20 μl of Bac-FLPo and 20 μl of substrate baculovirus to 60 μl of Sf-9 medium. Mix the 100 μl of virus mixture with 400 μl of surrounding solution. For mock transduction as control, simply mix 100 μl of Sf-9 medium with 400 μl of surrounding solution.

    • viii For each well, add the required volume of Bac-FLPo and substrate baculovirus into Sf-9 medium to a total volume of 100 μl. Next, mix the 100 μl of virus solution with 400 μl of surrounding solution (NaHCO₃-free rASC medium).

    • ix Discard rASC medium and wash the cells twice with PBS (1 ml per well). Remove the PBS.

    • x Initiate the transduction by adding the mixed virus solution (500 μl) into each well.

    • xi Put the six-well plate on a rocking plate. Continue the transduction process by gently shaking the plate (10 times/min) for 6 h at room temperature.

    • xii After 6 h of transduction, remove the virus solution and add rASC medium (containing 3 mM sodium butyrate). Incubate the mixture at 37 °C for 24 h.

    • xiii Day 5 (−6). At 1 dpt, replace the sodium butyrate-containing medium with fresh rASC medium.

      Troubleshooting

    Critical Step

    Here we describe the co-transduction of rASCs cultured in a six-well plate with the FLPo/Frt-based hybrid baculovirus system (Bac-FLPo and its substrate baculovirus) as an example. This procedure starts from the thawing of the primary rASCs, isolated as described in Box 1.

    Timing 5–6 d

  • D Transduction of a rASC sheet

    • i Day 1. Seed rASCs (prepared as described in Step 57C(i–iii)) to a six-well plate (5 × 10⁵ cells per well) with 2 ml of rASC sheet fabrication medium. Keep the cells at 37 °C in a 5% CO₂ incubator for 2 d.

      Critical Step

      To facilitate the cell sheet formation, higher seeding cell density (5 × 10⁵ cells per well) is helpful. The 20% (vol/vol) of FBS in the fabrication medium also augments the secretion of extracellular matrix by rASCs to form the cell sheet.

    • ii Day 3. The rASC sheet should be visible. Remove the medium and wash the cell sheet gently with PBS (2 ml per well) twice.

    • iii Calculate the required virus volume.

    • iv For each well, add the required volume of baculovirus (e.g., Bac-CE) into Sf-9 medium to a total volume of 100 μl. Next, mix the 100 μl of virus solution with 400 μl of surrounding solution (NaHCO₃-free rASC medium).

    • v Initiate the transduction process by adding the virus solution to wells (500 μl per well). Put the six-well plate on a rocking plate. Continue the transduction process by gently shaking the plate (10 times per min) for 6 h at room temperature.

    • vi Terminate the transduction process by removing the virus solution.

    • vii Add the rASC sheet fabrication medium containing 3 mM sodium butyrate (2 ml per well) and incubate it at 37 °C for 15 h.

    • viii Day 4. Replace the medium with fresh rASC sheet fabrication medium and continue to incubate the cell sheet for 9 h.

    • ix Wash the cell sheet with PBS twice and statically incubate with 0.05% (wt/vol) trypsin-EDTA (0.5 ml per well) for 10 s.

      Critical Step

      The time for trypsin-EDTA treatment is crucial. Longer treatment time (e.g., 2 min) may dissociate the cell sheet. For rASC sheet, 10 s is sufficient to detach the cell sheet without compromising the sheet integrity.

    • x Remove trypsin-EDTA and wash the rASC sheet with PBS twice.

    • xi Remove residual PBS and replenish with 2 ml of PBS. Gently shake the six-well plate by hand until the transparent rASC sheet spontaneously detaches from the well. In general, gentle shaking for <1 min is sufficient to detach the cell sheet.

      Critical Step

      To quantify the transduction efficiency, the cell sheet can be dissociated by prolonging the trypsin-EDTA treatment time to 5 min. The dissociated cells are analyzed by flow cytometry.

    Timing 4 d

Troubleshooting

Troubleshooting advice can be found in Table 2.

Table 2 Troubleshooting table.

Timing

Steps 1–33, generation of recombinant bacmid: 5 d

Steps 34–44, production of recombinant baculovirus (P0): 5–7 d

Steps 45–56, amplification of recombinant baculovirus (P2): 10–12 d

Step 57, transduction of mammalian cells with baculovirus: 3–6 d

Box 1, titration of recombinant baculovirus: 12–15 d

Box 2, isolation and culture of rASCs: 21–23 d

Anticipated results

The transduction procedures in Step 57 option A yield high transduction efficiencies and transgene expression levels in a wide variety of mammalian cell lines (Fig. 4). However, iPS cells are less permissive to baculovirus transduction. At MOI 100, the transduction efficiency was ∼45% using option B (data not shown). For recombination efficiency analysis, the cells were co-transduced with the recombinase-expressing baculovirus (Bac-FLPo or Bac-Cre) and the substrate baculovirus Bac-ALF and analyzed by flow cytometry at 1 dpt. Co-transduction resulted in recombination efficiencies approaching 90%–95% in various cell lines (e.g., Huh-7, HeLa, BHK) and stem cells such as rASCs (Fig. 5)⁴⁵.

Figure 4: Analysis of transduction efficiency in mammalian cell lines transduced with an EGFP-expressing baculovirus (Bac-CE).

(a) Illustration of Bac-CE. (b) Fluorescence micrographs captured at 1 dpt. Magnification, 200×. Scale bars, 100 μm. (c) Transduction efficiency (TE) and total fluorescence intensity (FI). Huh-7, SNU-449, A549, HeLa and HEK293 cells were transduced with Bac-CE at MOI 100 and analyzed by flow cytometry. The data represent the means ± s.d. of three independent experiments.

Figure 5: Recombination efficiency in different cells.

(a) Illustration of Bac-ALF vector and recombined minicircle. Co-transduction of cells with the recombinase-expressing baculovirus and Bac-ALF results in the transgene excision and recombination to form an ∼2.2-kb minicircle and hence the placement of d2egfp downstream of the CMV promoter. Therefore, analysis of GFP⁺ cells yields recombination efficiency. (b) Comparison of Cre- and FLPo-mediated recombination in different mammalian cells. At 1 dpt, the d2EGFP expression was monitored with a fluorescence microscope. Scale bars, 100 μm. (c) Recombination efficiency analysis in different cells. HEK293, Huh-7, HeLa, BHK, RD, rASCs and hASCs were co-transduced with Bac-Cre or Bac-FLPo (MOI 50) and Bac-ALF (MOI 200). At 1 dpt, the cells were analyzed by flow cytometry. The data represent the means ± s.d. of three independent experiments. Panels b and c were adapted from ref. 45 under Creative Commons license 3.0.

rASCs were co-transduced with Bac-FLPo (MOI 100) and a substrate baculovirus (Bac-FCBW, MOI 100) encoding BMP-2 (a growth factor that stimulates osteogenic differentiation of ASCs), as described in Step 57C. The co-transduction enhanced and prolonged the BMP-2 expression to ∼21 d, which significantly exceeded the BMP-2 expression duration (∼7 d) conferred by a conventional baculovirus vector Bac-CB (Fig. 6)⁵⁴. In parallel, the co-transduced rASCs were collected at 1 dpt, seeded to scaffolds and implanted into critical-size (8 mm in diameter) calvarial defects in NZW rabbits⁵⁴. To compare the calvarial bone healing effects, two types of scaffolds were used: (i) gelatin sponge that stimulates calvarial bone healing and (ii) PLGA (poly(lactic-co-glycolic acid)) scaffold that does not promote calvarial bone healing. The co-transduced rASCs/gelatin constructs successfully filled ∼86% of the defect area and ∼61% of the defect volume at 12 weeks post transplantation, whereas the control cell/scaffold constructs (mock-transduced rASCs/gelatin and co-transduced rASCs/PLGA) failed to heal the defects (Fig. 7)⁵⁴.

Figure 6: Prolonged BMP-2 expression via hybrid baculovirus.

(a) Illustration of the BMP-2-expressing conventional baculovirus (Bac-CB) and hybrid baculovirus vectors (Bac-FCBW). (b) Time course of BMP-2 expression in rASCs. rASCs were mock-transduced, singly transduced with Bac-CB (MOI 100) or co-transduced with Bac-FLPo (MOI 50) and Bac-FCBW (MOI 100). The culture medium was collected at 1, 4, 7, 14, 21 and 28 dpt and analyzed by ELISA. The data represent the means ± s.d. of three independent experiments.

Figure 7: rASCs engineered with the hybrid baculovirus augment the healing of bone defects.

(a) Schematic illustration of experimental procedures including transduction, seeding and transplantation. rASCs were co-transduced with Bac-FLPo (MOI 100) and Bac-FCBW (MOI 100) and seeded into PLGA or gelatin sponges at 1 dpt, followed by transplantation into rabbit calvarial bone defects. (b) The maximum-intensity projection (MIP) images, microcomputed tomography (ìCT) and sagittal-view images obtained at 12 weeks post implantation. This experiment was performed on five animals, and representative data are presented showing that baculovirus-transduced rASCs/gelatin scaffolds (persistently expressing BMP-2) significantly improved calvarial bone healing in comparison with the controls (mock-transduced rASCs/gelatin and co-transduced rASCs/PLGA). Panel b is adapted from: The use of ASCs engineered to express BMP2 or TGF-âβ3 within scaffold constructs to promote calvarial bone repair, C.-Y. Lin et al., Biomaterials 34, 9401–9412, Copyright 2013, with permission from Elsevier (ref. 54).

To treat myocardial infarction (MI), rASC sheets were co-transduced with Bac-FLPo and a substrate vector expressing VEGF (a growth factor stimulating angiogenesis), as described in Step 57D. Epicardial implantation of the VEGF-expressing rASC sheet to rabbit MI models reduced the infarct size, improved cardiac functions, prevented myocardial wall thinning, suppressed myocardium fibrosis and enhanced blood vessel formation, implicating the potential of baculovirus-engineered, VEGF-expressing rASC sheet for future MI treatment²². Thus, these examples of the types of results optioned using baculovirus transduction demonstrate the potential of the hybrid baculovirus system and transduction protocol for stem cell engineering and tissue regeneration.