Due to different research purposes, different species and tissues have been selected to be used for isolating and culturing fibroblasts for experiments. Some studies have pointed out that the number of passages, confluence state, growth rate, and division phase of fibroblasts before they are used for nucleofection can affect nucleofection efficiency. Although opinions differ on the optimal number of passages for fibroblasts, most studies agree that cells should be kept in a low-passage exponential growth phase prior to transfection. Reaching high confluency should be avoided, since cells in a high confluence state are more resistant to reprogramming. In addition, the composition of the fibroblast culture medium was also indicated to correlate with the final nucleofection efficiency by affecting the proliferation rate of fibroblasts. So far, an overview over experimental design and statistical data on specific effects of these factors on fibroblasts nucleofection efficiency is missing.

Fibroblast extraction

Fibroblasts have been successfully extracted from diverse species and tissues for research by enzymatic and explant culture methods. The enzymatic method mostly refers to the use of collagenase, dispase, hyaluronidase or trypsin to digest the chopped tissue specimens. The typical explant method consists of cutting the sample into tissue fragments of about 0.5 mm in length, planting them in a petri dish with culture medium, and letting the cells grow out of the sample. For example, we have successfully isolated fibroblasts from the dermis of the skin obtained from the back of Lewis inbred rats by the enzymatic method in previous studies. In these experiments, the first 3 passages of the cultured cells were used for subsequent experiments [11, 13]. Some studies have also successfully extracted fibroblasts from the dermis of human skin by enzymatic and explant culture methods [23,24,25,26,27,28]. Skrzyszowska et al. and Ko et al. minced the ear skin of 6-month-old and 10-day-old sows, respectively, as tissue explants to culture monolayers of adherent fibroblasts [29, 30]. Skrzyszowska et al. chose to culture fibroblasts for more than 3 passages before using them in subsequent experiments, whereas Jacobsen et al. extracted primary cells and put them into use immediately, or the cells were firstly cryopreserved and then recovered before putting into use. In a study by Zanin et al., sciatic nerves of 8–10 week old rats were taken out and cut into 1 mm length segments for use as explants to isolate fibroblasts [31]. Miki et al. removed islets from brain-dead donors, selected healthy parts and isolated human fibroblasts [32]. Fibroblasts can also be extracted from rat connective tissue, C57BL/6 mouse embryos, zebrafish embryos and Murrah buffalo embryos by enzymatic digestion or explant culture [33,34,35,36]. Furthermore, fibroblast cell lines are also often studied as nucleofection targets, such as the human fibroblast cells Hs27 [37], human skin fibroblast cell line CCD-1079Sk [38], human foreskin fibroblast cell line HFF-1 [27], mouse embryonic fibroblast cell line NIH3T3 [39].

Fibroblast passage and culture

Notwithstanding some studies emphasize the necessity to use cells within three to five passages in the nucleofection process [15], some studies recommend that the number of passages needs to be more than three [29]. However, from the above studies, it is impossible to directly observe the effect of using primary cells with different passages or cell lines with more passages on the subsequent nucleofection process. Perhaps the most suitable fibroblast type or passage number for nucleofection can be explored by the same transfection method for primary fibroblasts or cell lines from different sources and passages. Moreover, Kime et al. pointed out that during the cell culture process before transfection, the cells should be kept at a low passage, maintained in the exponential growth stage, and prevented from reaching high confluence, because when cells proliferate to a highly confluent state, their growth rate decreases while their resistance to reprogramming increases, which may be detrimental to the subsequent nucleofection process [40]. Another research team also expressed a similar view, believing that when fibroblasts grow to 80–90% confluence, they should be passaged at a ratio of 1:5 [40]. Keeping the fibroblasts in the exponential growth stage, where they are actively dividing, allows to make use of the nuclear membrane rupture during the nuclear division, which is conducive to gene reprogramming. Another important aspect is nutrition of the cells. Most studies use Dulbecco's Modified Eagle Medium (DMEM) containing 10–20% fetal calf serum (FCS), 1%-2% non-essential amino acids, 2-4 mM glutamine, 1–1.5% b-mercaptoethanol and 1% penicillin–streptomycin to incubate skin-derived fibroblasts in 37 °C 5% CO2 humidified incubators. Some of these studies added basic fibroblast growth factor (bFGF) to the culture medium, but effects on subsequent transfection were not observed. Only Kuebler et al. and Teklemariam et al. chose Iscove's Modified Dubecco's Medium (IMDM) to culture fibroblasts [14, 24], IMDM contains higher concentrations of nutrients and is suitable for high-density cell culture, where fibroblasts can be harvested at higher yields than in DMEM. However, considering the main points of pre-transfection cell culture (maintaining in the exponential growth phase) as mentioned by Kime et al., the fibroblasts in IMDM can proliferate too fast, which may be inappropriate to control their quantity before transfection. The isolated fibroblasts can be stained with phalloidin and DAPI, respectively, to stain the cytoskeleton and nucleus for observation of cell morphology under a fluorescence microscope. In addition, the identification and characterization of fibroblasts can be accomplished by fluorescent staining with antibodies against various markers, such as human fibroblast surface markers Thy-1, tubulin, vimentin, cytokeratin-18 [15, 36, 41] or prolyl 4-hydroxylase beta subunit (P4Hβ) [13].

Fibroblast nucleofectionPre-nucleofection

There are a few publications reporting that the subsequent nucleofection process is significantly affected by different sources of fibroblasts. Some studies mentioned that primary fibroblasts and NIH3T3 cells are difficult to transfect [42,43,44], but in many other studies the transfection efficiency of these cells has been satisfactory. It can be achieved by using cells with lower passage numbers and controlling the confluency of the cells (40%-80%) in the culture dishes before transfection as described above to maintain cell viability and mitotic activity [15].

Cell contamination often severely affects transfection outcomes [45]. Bacterial and fungal contamination of cells is usually easily detected during cell culture due to obvious medium turbidity. The main exemption to this rule is contamination with mycoplasma, which may not render the medium turbid, but in most cases, such pathological changes of mycoplasma-contaminated cells are relatively mild, mainly cell proliferation is slowed down. Phase contrast microscopy, electron microscopy, and DNA fluorescence staining can be used to detect the presence of mycoplasma. Once mycoplasma infection is found, mycoplasma-sensitive antibiotics should be added or experimental cells should be replaced.

NucleofectionSelection of nucleofection protocol and improvement of transfection solution

Nucleofection is an electroporation-based transfection modality with a variety of cell-specific transfection buffers and different programs that control voltage, frequency, and pulse duration. Skrzyszowska et al. used Sacl enzyme to cut the constructed plasmid DNA into linear conformation. They added fibroblast nucleofection buffer for nucleofection of pig fibroblasts, wherein the U-20 procedure was used for pig fetal fibroblasts, U-23 procedure was used for porcine adult dermal fibroblasts. In their work, they only give numbers for different protocols (e.g. U20, U23), while treating the exact procedure as a trade secret. The transferred target sequence carried an enhanced green fluorescent protein (eGFP) gene. This allowed to detect transfection efficiency of the gene by observing the fluorescence intensity of eGFP [29]. Ko et al. carried out nucleofection of porcine fibroblasts using the U-23 procedure. They reported a cell viability of up to 87.9%, and transfection efficiency of 80.8%. This was much higher than cell viability after lipofection (62.0–77.7%), which also had a very low transfection efficiency of 6.1–6.3% [30]. Mehta et al. nucleofected Murrah buffalo fetal fibroblasts using the EN-150 procedure, and the cell viability (53.8 ± 4.2%) and transfection efficiency (73.6 ± 1.4%) were both higher than those using Fugene HD (nucleofection efficiency: 50.4 ± 1.7%, cell viability: 24.6 ± 2.9%) and Lipofectamine 2000 (nucleofection efficiency: 71 ± 1.2%, cell viability: 30.4 ± 3.1%) lipofection [41]. Another study tested the transfection efficiency of 30 different procedures of nucleofection using porcine and rabbit embryonic fibroblasts, of which U-20 was the most efficient for porcine embryonic fibroblasts with a transfection efficiency of 90% and the cells toxicity was only 5%. U-23 was the most effective for rabbit embryonic fibroblasts with a transfection efficiency of 38%. Rabbit embryonic fibroblasts seem to be more difficult to transfect than porcine-derived fibroblasts. This study also tested the transfection efficiency of various chemical media transfection methods, among which the transfection efficiency of Effectene was 18%, Lipofectamine 2000 was 28%, Lipofectamine Plus was 20%, and polyethyleneimine was 32%, which were all significantly lower than the nucleofection efficiency of porcine embryonic fibroblasts, and even lower than the nucleofection efficiency of difficult-to-transfect rabbit-derived cells [46]. Other studies confirmed in human, porcine and mouse fibroblasts that nucleofection is much more efficient than conventional electroporation and lipofection [19, 28, 47, 48].

Previous studies have compared and found that the cuvettes provided by the manufacturer of the Nucleofector device and cuvettes from Eppendorf had no significant difference in the nucleofection process. Under certain circumstances, the transfection solution provided by the manufacturer could even be improved [13]. It was found that using the U-30 procedure for rat fibroblasts and replacing the nucleofection buffer with DMEM supplemented with 10% FCS had the highest transfection efficiency (about 85%) without affecting the growth and proliferation activity of the cells, which was a significant improvement over the transfection efficiency of standard fibroblast nucleofection buffer which only reached 68% in this application. Using the U-24 procedure for human dermal fibroblasts, but replacing the nucleofection buffer with DMEM supplemented with 10% FCS, the transfection efficiency (around 57%) was below that of the standard method of nucleofection (around 79%), but after replacing the standard transfection solution with ITS liquid medium supplement, the transfection efficiency was comparable to the standard solution (about 83%). As Eppendorf cuvettes, DMEM with FCS, and ITS liquid medium supplement are relatively inexpensive, a more economical and efficient nucleofection method was developed [11, 13].

Stability of gene integration and DNA damage response

Skrzyszowska et al. compared cell viability and proliferative activity after nucleofection and lipofection, both of which were higher in the nucleofection group. However, in PCR screening results, only in 1 of the 5 analyzed groups of nucleofection, the gene was integrated into the nuclear genome, while the transgenes of 3 of the 5 analyzed groups of lipofection were successfully integrated [29]. Therefore, although the transfection efficiency of nucleofection technology is high, it may have disadvantages in the stability of the integration process of the transgene into the nuclear genome. At the same time, it cannot be ruled out that the target gene construct in this experiment was resulted in the failure of the transgene or the loss of the transgene during the cell cloning process after nucleofection. On the other hand, in the study of Zanin et al., amazing performance of nucleofection in terms of high efficiency and long-term stability was reported using the T-16 procedure to nucleotransfect primary fibroblasts isolated from rat sciatic nerves. The transfection efficiency was much higher than that of lipofection, and the continuous expression duration of the target gene reached 30 weeks, which was also much longer than that of lipofection and even the target gene expression duration in lentiviral transfection in their study [31]. Although nucleofection has the advantages of high efficiency and low cytotoxicity, Huerfano et al. found that the use of U-30 procedure in NIH3T3 cells and mouse embryonic fibroblasts nucleofected with different target genes elicited a strong inducible type I interferon (IFN) response and DNA damage response (DDR). When the same cells were transfected with the same plasmid by means of a cationic polymer (Turbofect), the levels of IFN and DDR were significantly lower than those by nucleofection [39]. The commercial description of nucleofection claims that DNA is "directly delivered" into the nucleus, but Huerfano et al. considered that this "direct delivery" is essentially just the faster transfer of DNA from the cell membrane to the nucleus. The faster transfection rate of nucleofection is also reflected in other studies. Compared with gene expression that starts 24 h after lipofection, nucleofection takes only 3 h [23]. Although not demonstrated, in addition to eliciting a higher degree of IFN, Huerfano et al. observed that nucleofection of different plasmids caused similar inhibition of cell proliferation, which is rarely reported in other studies. Interferons can modulate some cellular physiological and pathological behaviors by participating in many cell signaling cascades. In many studies involving nucleofection, it has not been considered that this process may cause side effects of excessive IFN activation, which may have some influence on the results of these studies. It is speculated that this reaction is caused by the recognition of DNA by cytoplasmic DNA sensors when DNA is delivered in the cytoplasm, during the "rapid transfer" of DNA from the cell membrane to the nucleus. It is recognized by DNA sensors as fracture of DNA and further activates the DDR, another hypothesis is that the elevated ROS levels observed during nucleofection induce IFN responses as well as DDR. Unfortunately, the specific mechanism of DDR and IFN induced by nucleofection has not been verified and elucidated, and further exploration is needed. Kime et al. mentioned in a method guide that the addition of nucleofection solution and cell suspension in strict accordance with the dosage in the instructions, the control of the number of pre-transfected cells, and the control of the transfection operation time are all closely related to the success of nucleofection, the transfection efficiency and cytotoxicity [40].

The implementation conditions and transfection results of nucleofection in related studies are summarized in Table 1.

Table 1 The implementation conditions and transfection results of nucleofection in related studies

In order to more intuitively present the relationship between the sources of fibroblasts, the nucleofection procedures used in each study with the nucleofection efficiency and cell viability in Table 1, we drew a column-scatter chart (Fig. 2).

Fig. 2

Gray bar represents transfection efficiency, orange dot represents cell viability, the groups with no cell viability value indicate that the cell viability after nucleofection is not mentioned in the original papers. The corresponding references are marked below the program names

Post-nucleofection

The composition of the nucleofection standard solution is unreported, and may have a certain negative impact on cell survival and proliferation. Most studies stated that the cell viability after nucleofection is higher than other chemical or physical transfection methods, but it is always lower than the viability of untransfected cells. Thus, some researchers recommend that 24 h after nucleofection, the standard solution of nucleofection should be replaced with DMEM, IMDM or other inducible medium according to different experimental purposes [33, 50]. Considering the high efficiency of nucleofection (the target gene starts to translate from 3 h after transfection), Badakov et al. replaced the transfection reagent with culture medium 6 h after nucleofection [35]. Many studies use the pmaxGFP plasmid as a vector for nucleofection experiments. After transfection, flow cytometry or fluorescence microscopy can be used to quantify the transfection efficiency by GFP fluorescence [13, 14, 20, 24, 28, 29, 36, 38, 41, 46, 51]. The transfection efficiency can also be observed and detected with fluorescence microscopy by fluorescent staining against specific marker proteins encoded by the target genes [32, 50, 52]. Using qRT-PCR, Western Blot or ELISA to detect the expression of the transfected target genes can also partially reflect the transfection status, but the expression of the genes is affected by many factors, so it can only be regarded as a partial reference for the success of the transfection [23, 27, 37]. There are some studies that did not use plasmids but siRNA for nuclear transfection of fibroblasts, and then detected the expression of the target genes of the siRNA through qRT-PRC and Western Blot methods [25]. Some researchers aimed to generate human induced pluripotent stem cells through nucleofection from fibroblasts. This process may take 21 days or more and they considered that fibroblasts will become senescent after 14 days of nucleofection, which would affect the experimental results. Therefore, they performed a second nucleofection on days 2–7. Surprisingly, the cells that underwent the second round of nucleofection died in large numbers, even though they were able to obtain pluripotent marker-positive target cells. Some of the selected cell clones were found stopping proliferation and turning to apoptosis within 2 weeks. Perhaps it should be considered whether there is genetic damage to the cells by secondary nucleofection, but this is not yet clarified [27]. The study by Huerfano et al. confirmed that nucleofection does cause IFN and DDR. The content of reactive oxygen species (ROS) in cells undergoing nucleofection increases, and the level of oxidative stress elevates [39], which may cause DNA damage, thereby inhibiting the normal physiological functions of cells. As mentioned earlier, modification of the nucleofection standard solution, or early replacement of the nucleofection solution with culture medium may decrease the stress and reduce this damage to the cells.