Lipid-laden hepatocytes are inherent to liver regeneration but are usually lost upon density-gradient centrifugation. Here, we present an optimized cell isolation protocol that retains steatotic hepatocytes, yielding representative populations of regenerating hepatocytes after partial hepatectomy in mice.
Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream single-cell applications is challenging. A marked accumulation of lipids within regenerating hepatocytes is observed during the first 2 days of normal liver regeneration in mice. This so-called transient regeneration-associated steatosis (TRAS) is temporary but partially overlaps the major proliferative phase. Density-gradient purification is the backbone of most existing protocols for the isolation of primary hepatocytes. As gradient purification relies on the density and size of cells, it separates non-steatotic from steatotic hepatocyte populations. Therefore, fatty hepatocytes often are lost, yielding non-representative hepatocyte fractions.
The presented protocol describes an easy and reliable method for the in vivo isolation of regenerating hepatocytes regardless of their lipid content. Hepatocytes from male C57BL/6 mice are isolated 24-48 h after hepatectomy by a classic two-step collagenase perfusion approach. A standard peristaltic pump drives the warmed solutions via the catheterized inferior vena cava into the remnant, using a retrograde perfusion technique with outflow through the portal vein. Hepatocytes are dissociated by collagenase for their release from the Glisson’s capsule. After washing and careful centrifugation, the hepatocytes can be used for any downstream analyses. In conclusion, this paper describes a straightforward and reproducible technique for the isolation of a representative population of regenerating hepatocytes after partial hepatectomy in mice. The method may also aid the study of fatty liver disease.
The liver can regenerate itself even after major tissue loss. This unique regenerative capacity is explicitly illustrated by the experimental model of partial (70%) hepatectomy, first described in rats by Higgins and Anderson in 19311. In this model, 70% of the liver is surgically removed from animals by clipping off larger liver lobes. The remaining lobes then grow through compensatory hypertrophy to restore the original liver mass within about 1 week after surgery, albeit without restoration of the original liver architecture2,3. Additional hepatectomies with varying amounts of tissue removal have been developed, such as 86%-extended hepatectomy where the liver remnant is too small to recover, eventually leading to posthepatectomy liver failure (PHLF) and subsequent death in 30%-50% of the animals4,5,6. These models enable the study of normal and failed liver regeneration, depending on the amount of resected tissue (Figure 1).
Although mouse models of hepatectomies have been used successfully for many years, only recently have more advanced analytical methods allowed for a deeper insight at the single-cell level. For most of these methods, however, the presence of individual hepatocytes is a basic prerequisite. Most protocols for the isolation of primary hepatocytes are based on a two-step collagenase perfusion technique and subsequent density-gradient purification to separate viable hepatocytes from debris and non-parenchymal, as well as dead cells7,8,9. This method was first described by Berry and Friend in 196910 and adapted by Seglen and colleagues in 197211,12. However, as gradient centrifugation relies on the density and size of cells, lipid-laden hepatocytes are often lost during standard purification. While such loss may be negligible for many research questions, it is a crucial aspect for early liver regeneration. During the first 2 days, hepatocytes within the regenerating mouse liver accumulate lipids, thereby growing in size and dipping in density. This transient regeneration-associated steatosis (TRAS) serves to provide regenerative fuel and is temporary, but partially overlaps the major proliferative phase and is unevenly distributed within the liver lobules – the functional units of the liver13,14. After extended 86%-hepatectomy, however, TRAS also occurs but persists, because regeneration is stalled and lipids are not being oxidized14. Therefore, gradient-purification of hepatocytes following 70%- or 86%-hepatectomies will yield non-representative fractions, as most lipid-laden hepatocytes are lost due to their low density15.
In this modified isolation protocol, hepatocytes from C57BL/6 mice are isolated 24-48 h after hepatectomy by a classic two-step collagenase perfusion approach. Usually, cannulation and perfusion of the remnant for cell isolation are done via the portal vein. However, portovascular resistance in small remnants left after major resection is high16, and thus perfusion is delicate. Because the vena cava remains unaffected by hepatectomies, perfusion can be easily performed in the retrograde direction via cannulation of the vena cava. A standard peristaltic pump drives the warmed solutions via the catheterized inferior vena cava into the liver remnant, using retrograde perfusion with outflow through the portal vein (Supplementary Figure S1). Hepatocytes are dissociated by collagenases and released from the Glisson's capsule. After washing and careful processing of viable hepatocytes by stepwise isolation using a low-speed centrifugation approach, the hepatocytes can be used for any downstream analyses.
All animal experiments were in accordance with Swiss Federal Animal Regulations and approved by the Veterinary Office of Zurich (n° 007/2017, 156/2019) assuring human care. Male C57BL/6 mice aged 10-12 weeks were kept on a 12 h day/night cycle with free access to food and water. Each experimental group consisted of six to eight animals. See the Table of Materials for details related to all materials, equipment, and reagents used in this protocol.
1. Partial hepatectomy in mice
Figure 1: Standard (70%) and extended (86%) hepatectomy in mice. (A) The five mouse liver lobes and their respective contributions to the total liver weight. (B) Schematic illustration of 70%-hepatectomy in mice. The dark lobes represent the future liver remnant. (C) Schematic illustration of 86%-hepatectomy in mice. The dark lobes represent the future liver remnant. (D) Precise volume of resected tissue post 70%- and 86%-hepatectomy. (E) Mouse abdomen immediately after 70%-hepatectomy; (F) mouse abdomen immediately (left) and 48 h (right) after 86%-hepatectomy. Note the pale color of the steatotic remnant (white arrow). n = 6-7/group. Abbreviations: sHx = standard hepatectomy; eHx = extended hepatectomy; LW = liver weight. Please click here to view a larger version of this figure.
2. Preparation of the perfusion solutions
Table 1: Solutions and buffers used for the digestion and purification of hepatocytes. Please click here to download this Table.
3. Preparation of perfusion equipment
4. Cannulation and perfusion
Figure 3: Perfusion process from cannulation to digestion. (A) Anatomy of the mouse liver with the inferior vena cava (white arrow) and the portal vein (yellow arrow). (B) Cannulation of the inferior vena cava. The cannula is secured with a ligature (white arrow), and the location of the outflow through the opened portal vein is marked (not clamped) with a micro vessel clamp. (C) Note the appearance of patchy structures before the perfusion buffer has cleared the liver from all remaining blood (white arrow). The skin is incised (yellow arrow) and a cotton swab is placed to ensure drainage of blood and perfusion fluid. Intermittent clamping can be performed with a vascular clamp or tweezers. (D) The liver should be cleared of all blood (*). After the collagenase-containing digestion buffer has entered the liver, it will no longer relax after clamping and the liver lobes will increase in size. (E) After a while, a bubbly appearance on the surface of the liver can be observed (*). Please click here to view a larger version of this figure.
5. Digestion
6. Preparation of the liver
7. Hepatocyte extraction
Figure 4: Purification by gentle centrifugation. (A) Liver homogenate left after the extraction step. (B) Microscopic view (20x magnification) of the homogenate; note the marked contamination with debris. (C) Purification centrifugation steps and (D) microscopic views of the supernatants to be discarded. (E) Microscopic view of the purified hepatocyte fraction. Scale bars = 100 µm. Please click here to view a larger version of this figure.
8. Hepatocyte isolation
9. Preparation of the isolated hepatocytes for flow cytometry
10. Analyzing hepatocytes with flow cytometry
TRAS peaks at 16 h post hepatectomy and gradually vanishes 32-48 h after standard hepatectomy, but persists beyond 48 h after extended hepatectomy. Macroscopically, TRAS is readily visible as a pale complexion of the liver remnant (Figure 1F) and can be observed in hepatectomized mice between 16 h and 48 h after surgery.
The estimated final yield is 10-15 × 106 hepatocytes after 70%-hepatectomy and 4-9 × 106 after extended 86%-hepatectomy in mice, with a mean final viability of 78% and 65%, respectively. This corresponds to the expected percentage compared to a total yield of 50-70 × 106 from a whole mouse liver (Figure 6A,C). After partial hepatectomies, hepatocytes show an increased cell size compared to normal livers, corresponding to their increased lipid-content (Figure 6B). The gating strategy is shown in Supplementary Figure S4.
Figure 6: Hepatocyte yield, cell size, and viability. (A) Cell count of one whole mouse liver and of remnants 24 h post 70%- and 86%-hepatectomy. (B) Calculated cell volume of isolated hepatocytes. Note the marked increase in cell size 24 h post hepatectomy, for both 70% and 86%. (C) Cell viability of isolated hepatocytes after each step of the purification process. Viability was measured by flow cytometry using Alexa Fluor 488 Zombie green viability dye. Percentages are taken from all hepatocyte singlets. For the gating strategy, see Supplementary Figure S6 and Supplementary Figure S4. Error bars refer to standard deviations with n = 6-8/group. Abbreviations: sHx = standard hepatectomy; eHx = extended hepatectomy; w/o resection = sham surgery. Please click here to view a larger version of this figure.
An increased lipid content in murine hepatocytes 24 h after partial hepatectomy can be observed by an increase in granularity (Figure 7; for gating strategy, see Supplementary Figure S5) or directly observed by the presence of lipid vesicles inside enlarged hepatocytes (Figure 8 and Supplementary File 1).
Figure 7: Increased granularity and cell size measured by flow cytometry. (A) Increased granularity among isolated hepatocytes 24 h post hepatectomy as an indirect marker for the presence of lipid vesicles. (B) Histogram showing SSC. (C) The percentages represent the fraction of cells with high cytoplasmic granular intensity, measured by the SSC signal. Error bars refer to standard deviations with n = 6-8/group. Abbreviations: sHx = standard hepatectomy; eHx = extended hepatectomy; w/o resection = sham surgery; FSC = forward scatter signal; SSC = side scatter. Please click here to view a larger version of this figure.
Figure 8: Sudan IV staining as a marker of fat content of isolated fresh hepatocytes. (A) Hepatocytes from a fresh whole mouse liver without prior surgery. Increased size and fat content of hepatocytes 24 h (B) post 70%-hepatectomy and (C) 86%-hepatectomy. Note the concomitant presence of both larger, steatotic hepatocytes and smaller, non-steatotic hepatocytes. Scale bars = 30 µm (B–D). Error bars refer to standard deviations with n = 6-8/group. Abbreviations: sHx = standard hepatectomy; eHx = extended hepatectomy; w/o resection = sham surgery. Please click here to view a larger version of this figure.
A very low percentage of immune and non-parenchymal cells remain in the final suspension. Further purification is achieved by FACS or MACS, if desired. Negative selection of CD31+ and CD45+ cells by flow cytometry is used to demonstrate and quantify the non-parenchymal cells (Figure 5).
Figure 5: Flow cytometry gating strategy and selection of CD31– and CD45– parenchymal cells. (A) Gating chart with all events recorded; consider the relatively large size of hepatocytes and use adjusted voltages. Do not go higher than 350 V for FSC and 220 V for SSC. (B) Singlet gating. (C) Hepatocytes are selected by gating CD31– (endothelial marker) and CD45– (immune marker) cells. This selection can be performed in a fluorescence-activated cell sorter to select parenchymal cells for further downstream analyses, if needed; n = 4-5/group. Abbreviations: FSC = forward scatter; SSC = side scatter. Please click here to view a larger version of this figure.
To demonstrate the efficacy of the modified protocol in retaining steatotic cells, hepatocytes were isolated using the classic density-gradient approach7 (Figure 9A). Unlike with the modified procedure (Figure 9B), a fatty cell layer was clearly visible on top of the density gradient. Analysis of cells confirmed a gross absence of lipid-laden hepatocytes in the pellet following density-gradient centrifugation. In contrast, cells from the fatty layer were enriched with steatotic hepatocytes and a substantial fraction of non-parenchymal and dead cells (Figure 9A). Thus, classic isolation deprives the harvest of steatotic cells, while this modified protocol omitting the density gradient retains lipid-laden subpopulations, enabling the unbiased exploration of liver regeneration without skewing toward lean hepatocytes.
Figure 9: Yields of steatotic hepatocytes following the classic density-gradient isolation protocol compared to the improved protocol. (A) With the classic density-gradient purification method (final concentration of 90% density-gradient solution in phosphate-buffered saline), dead hepatocytes are collected in a cell layer on top of the density-gradient solution. (B) This layer does not only consist of non-parenchymal cells and non-viable hepatocytes but also of viable, large, fatty hepatocytes. (C) The pellet obtained following density-gradient centrifugation contains lean hepatocytes of smaller size and is mostly devoid of steatotic cells. This is the "pure" fraction that is isolated with the classic protocol. (D) With the improved protocol, the lipid-filled hepatocytes are not lost and all hepatocytes are pelleted. The supernatant (E) consists mostly of dead and fragmented hepatocytes and non-parenchymal cells, while the pellet (F) is a mixture of hepatocytes of variable size and lipid-content. Scale bars = 100 µm. Please click here to view a larger version of this figure.
Supplementary File 1: Supplementary protocols. (A) Lipid staining with Sudan IV; (B) standard and extended hepatectomy in mice. Please click here to download this File.
Supplementary Figure S1: Overview on the perfusion setup. (1) Cannulate the inferior vena cava. (2) Perfuse the liver with warm perfusion buffer to chelate calcium. (3) Warm the digestion buffer with collagenases and Ca2+ to dissociate cells in vivo. Remove the liver and (4) store in ice-cold preservation buffer (maximum 30 min). Please click here to download this File.
Supplementary Figure S2: Perfusion setup. (A) Perfusion table with an isoflurane nozzle, red light heating lamp, and perfusion tube. A heavy object can be placed underneath the tube to stabilize it and reduce the risk of displacement. (B) During the first few minutes of the perfusion, some blood will outflow through the portal vein and pool in the open abdominal cavity. (C) The abdominal wall is incised on one side to drain the blood. A cotton swab facilitates the drainage. Please click here to download this File.
Supplementary Figure S3: Glisson's capsule. (A) The Glisson's capsule is ruptured with fine-tip tweezers in a few locations along the liver surface, and the liver cells are released by gently shaking the capsule. (B) The liver should tear apart easily. (C) The empty liver sac (Glisson's capsule) with released hepatocytes in suspension. Please click here to download this File.
Supplementary Figure S4: Flow cytometry gating strategy for viability screening. (A) All events were recorded. (B) Singlet gating. (C) Live/dead staining with Alexa Fluor 488 Zombie green viability dye. Please click here to download this File.
Supplementary Figure S5: Flow cytometry gating strategy for granularity measurement. (A) All events were recorded. (B) Singlet gating. (C) Dot plot of side scatter (SSC; x axis) versus forward scatter (FSC; y axis) to analyze the levels of granularity. Please click here to download this File.
Supplementary Figure S6: Hepatocyte purification. (A) Cell pellet after the first centrifugation step; note the fatty layer on top of the medium. (B) Hepatocyte pellet after the second round of centrifugation. (C) Purified hepatocytes at the end of the purification process. Please click here to download this File.
The published protocol provides a reliable and straightforward method to isolate a high yield of normal and steatotic murine hepatocytes for single-cell downstream analyses or bulk analysis of cells following FACS sorting. The distinct advantage over density-gradient purification is that the cellular lipid content has essentially no impact on the effective yield of hepatocytes. Thus, the fraction of steatotic hepatocytes will be retained and included in downstream analyses. This is not only crucial for the study of steatogenic liver diseases but also paramount for any analysis of processes following major hepatectomies, where hepatocytes display a temporally and spatially dynamic steatosis within the first 2-3 days of regeneration. Even though (single-cell) analyses of isolated hepatocytes have already been performed after hepatectomies19,20,21, it must be assumed that the cell population analyzed was, at least partially, deprived of lipid-laden hepatocytes and the results, therefore, were not fully representative and skewed toward lean hepatocytes.
Currently, the function of TRAS is not fully clarified. For example, the spatial differences in lipid content within liver lobules remain ill-understood. Therefore, a method is needed that allows quantitative and qualitative detection without precisely excluding these cells from analyses (e.g., for single-cell transcriptomics, flow cytometry, and metabolomics).
Overall, hepatocyte yield with this improved protocol is markedly superior over other methods for the isolation of fat-containing hepatocytes (e.g., from mice with nonalcoholic steatohepatitis, NASH) using density-gradient purification15. However, cell yield, viability, and fat content can vary between preparations. Several factors seem to be responsible.
First, different batches of collagenase or collagenase blends will vary in their enzymatic activity; however, the lot-to-lot differences are not as critical as with traditional collagenases. Nevertheless, adjusting the working concentration may be necessary. The differences can be further minimized by proper storage until use (dry lyophilizates and rehydrated stock solutions at -15 to -25 °C) and by avoiding repetitive freeze-thaw cycles, but cannot be entirely eliminated. Therefore, it is recommended to use only collagenases of a specific batch for a given experimental series. In addition, the enzymatic activity depends on the temperature at which the digestion buffer reaches the liver, with an optimal temperature between 35 °C and 37 °C for a blend of collagenases I and II22. Lower temperatures will reduce the enzymatic activity, while temperatures above 50 °C can lead to irreversible denaturation of the protein. Test this in advance and optimize the experimental conditions by adjusting the initial temperature of the digestion buffer, the length and diameter of the plastic tube, and the flow velocity. For example, a drop in buffer temperature of up to 10 °C within a tube length of 60 cm is to be expected. Correct the temperature by using warming pads and infrared lamps, if needed. Collagenases display their optimum digestion capacity at a pH of 7.4; therefore, it is advised to adjust the pH of the buffer solutions already at a working temperature of approximately 37 °C before use.
Second, it is paramount to flush the liver (or the whole mouse) sufficiently with perfusion buffer before starting the digestion process to eliminate potential inhibitors such as serum or albumin. Ideally, perfusion is started only after the systemic circulation has stopped, to ensure the digestion buffer moves only from the vena cava through the liver to the outflow via the opened portal vein. The superior vena cava can be closed with a small vascular clamp. This prevents contact with residual blood components/inhibitors. Another method of optimally flushing the liver is intermittent clamping of the portal vein. This should be done carefully and – once the digestion buffer has reached the liver – performed only once to avoid damaging the already digested cells.
Third, regenerating hepatocytes are sensitive, and experience has shown that they require shorter digestion times and more careful handling to avoid over-digestion and harm to the cells. To harvest the liver as gently as possible, it is recommended to grasp the hepatic hilus with a clamp and then, first cut the superior vena cava and free the liver from the connections to the vein and the diaphragm. Subsequently, the inferior vena cava and the portal vein can be cut, which enables mobilization of the liver and easily frees it from the gastrointestinal ligaments, the esophagus on one side, and the right kidney on the other side. In hepatectomized mice, it is possible to carefully grasp one of the ligatures from the median lobes to remove the liver. If an attempt is made to pull the liver out by force, the Glisson's capsule may rupture, and the isolated cells could get lost.
Finally, it is essential that the perfusion procedure and further processing of the hepatocytes are not interrupted or delayed, as this will immediately and inevitably affect viability. Ideally, the perfusion is performed in a team of at least two people and on one animal at a time. These requirements in time and manpower, however, are one of the limitations of this method, although this applies to alternative methods as well.
Another limitation is the final purity of the resulting cell suspension, as the advantage of not losing steatotic hepatocytes to a density gradient results in a small proportion of remaining non-parenchymal and/or immune cells. In the shown sample, the fraction of CD45+ (immune cells) and CD31+ (endothelial cells) is below 5%, and when selecting for cell size first during flow-cytometry, the percentages of CD45+ and CD31+ are 1.2% and 2.2%, respectively (Figure 5). For most applications, this level of purity is sufficient, but highly sensitive methods or the further use in primary cell cultures probably requires a higher degree. Numerous studies have used CD31 and CD45 as systemic markers to sort hepatic cell populations by MACS23,24 or FACS25.
This improved isolation method is ideally suited for the study of liver regeneration, particularly the early period that presents with significant amounts of steatotic hepatocytes. PHLF, featuring persisting steatosis, is a particularly relevant subject requiring research. Following extended hepatectomies leaving behind marginal remnants, PHLF can develop and indeed represents the most common cause of death due to liver surgery. Its pathobiology is only partially understood, and currently no treatment is available26. Given that PHLF significantly limits the application of liver surgery (such as for the cure of hepatic malignancies), exploring its causes and identifying potential measures is a clear medical need.
Need also exists for the study of diseases that share hepatic steatosis as a starting point. A prominent example is nonalcoholic fatty liver disease (NAFLD), which may progress to NASH and eventually to hepatocellular carcinoma (HCC)27. The spread of sedentary lifestyles in conjunction with the Western diet has led to a worldwide epidemic of NAFLD, and accordingly, HCC incidence is projected to rise28,29. Steatosis, in turn, is also tightly linked to obesity, the metabolic syndrome, and type-2-diabetes, all diseases with a rising incidence30. Therefore, steatosis represents an increasing burden for the current healthcare system, calling for effective means for its management. This improved two-step collagenase perfusion method enables the study of steatotic hepatocytes at the single-cell level, and hence should aid in exploring the causes and consequences of hepatocellular lipid accumulation.
This protocol presents an easy, fast, and reliable technique for the in vivo isolation of regenerating hepatocytes from mice after partial (70%) and extended (86%) hepatectomy. This approach does not rely on gradient-purification and can be used to investigate liver regeneration processes with the inclusion of lipid-laden hepatocytes that are usually lost during traditional isolation protocols. Moreover, this method can also be applied for the research of various liver diseases associated with steatotic changes and is not limited to the mouse species.
The authors have nothing to disclose.
This study was supported by the Swiss National Fond (project grant 310030_189262).
Reagents | |||
Alexa Fluor 488 Zombie green | BioLegend | 423111 | Amine-reactive viability dye |
Attane Isoflurane ad us. vet. 99.9% | Provet AG | QN01AB06 | CAUTION: needs ventilation |
EDTA solution | Sigma-Aldrich | E8008-100ML | – |
Ethanol | Sigma-Aldrich | V001229 | Dilute with water to 70% |
Fetal bovine serum (FCS) | Gibco | A5256701 | – |
Hanks' Balanced Salt Solution (HBSS), Ca2+, Mg2+, phenol red | Sigma-Aldrich | H9269-6x600ML | For digestion/preservation |
Hanks' Balanced Salt solution (HBSS), w/o Ca2+, w/o Mg2+, no phenol red | Sigma-Aldrich | H6648-6x500ML | For perfusion buffer |
HEPES solution, 1 M | Sigma-Aldrich | 83264-100ML-F | – |
Histoacryl tissue adhesive (butyl-2-cyanoacrylate) | B. Braun | 1050052 | For stabilization of cannulation site |
Hoechst 33258 Staining Dye Solution | Abcam | ab228550 | – |
Liberase Research Grade | Roche | 5401119001 | Lyophilized collagenases I/II |
NaCl 0.9% 500 mL Ecotainer | B. Braun | 123 | – |
Paralube Vet Ointment | Dechra | 17033-211-38 | – |
Phosphate buffered saline (PBS) | Gibco | A1286301 | – |
Sudan IV – Lipid staining | Sigma-Aldrich | V001423 | – |
Temgesic (Buprenorphine hydrochloride), Solution for Injection 0.3 mg/mL | Indivior Europe Ltd. | 345928 | Narcotics. Store securely. |
Trypan blue, 0.4%, sterile-filtered | Sigma-Aldrich | T8154 | For cell counting |
Williams’ Medium E | Sigma-Aldrich | W4128-500ML | – |
Materials | |||
25 mL serological pipette, Greiner Cellstar | Merck | P7865 | – |
50 mL Falcon tubes | TPP | – | – |
BD Neoflon, Pro IV Catheter 26 G | BD Falcon | 391349 | – |
Cell scraper, rotating blade width 25 mm | TPP | 99004 | – |
Falcon Cell Strainer 100 µm Nylon | BD Falcon | 352360 | – |
Fenestrated sterile surgical drape | – | – | Reusable cloth material |
Filling nozzle for size 16# tubing (ID 3.1 mm) | Drifton | FILLINGNOZZLE#16 | To go into the tubes |
Flow cytometry tubes, 5 mL | BD Falcon | 352008 | – |
Male Luer to Barb, Tubing ID 3.2 mm | Drifton | LM41 | Connection tube to syringe |
Petri dishes, 96 x 21 mm | TPP | 93100 | – |
Prolene 5-0 | Ethicon | 8614H | To retract the sternum |
Prolene 6-0 | Ethicon | 8695H | For skin suture |
Prolene 8-0 | Ethicon | EH7470E | Ligature gall bladder |
Tube 16#, WT 1.6 mm, ID 3.2 mm, OD 6.4 mm | Drifton | SC0374T | Perfusion tube |
Equipment | |||
BD LSRFortessa Cell Analyzer Flow Cytometer | BD | – | – |
Isis rodent shaver | Aesculap | GT421 | – |
Isofluran station | Provet | – | – |
Low-speed centrifuge – Scanspeed 416 | Labogene | – | – |
Neubauer-improved counting chamber | Marienfeld | – | – |
Oxygen concentrator – EverFlo | Philips | 1020007 | 0 – 5 L/min |
Pipetboy – Pipettor Turbo-Fix | TPP | 94700 | – |
Shenchen perfusion pump – YZ1515x | Shenchen | YZ1515x | – |
Surgical microscope – SZX9 | Olympus | – | – |
ThermoLux warming mat | Thermo Lux | – | – |
Vortex Genie 2, 2700 UpM | NeoLab | 7-0092 | – |
Water bath – Precision GP 02 | Thermo scientific | – | Adjust to 42 °C |