CN113046309B - Culture medium for suspension culture of brain organoids and application thereof - Google Patents

Abstract

The invention belongs to the field of biology, and particularly relates to a culture medium for suspension culture of brain organoids and application thereof. The culture medium comprises a first stage culture medium, a second stage culture medium, a third stage culture medium and a fourth stage culture medium; the culture medium comprises NEAA1% -2%, glumax1% -2%, mercap ethanol80-120nM; the first stage medium, the second stage medium and the third stage medium comprise polyvinyl alcohol. The invention successfully separates the mesenchymal stem cells from the brain organoids by using the culture medium, the early-stage pluripotent stem cells adopt a suspension culture method, the damage to cells is small, the yield is high, polyvinyl alcohol is added in the later-stage cell culture medium in an original way, and the whole system can stably and efficiently produce organoids; the obtained mesenchymal stem cells have application in preparing medicaments in the field of nerve repair, and particularly have wide application prospects in preparing medicaments for treating Alzheimer's disease.

Description

Culture medium for suspension culture of brain organoids and application thereof

Technical Field

The invention belongs to the field of biology, and particularly relates to a culture medium for suspension culture of brain organoids and application thereof.

Technical Field

Organoids (Organoids) are micro-organs with three-dimensional structures that are cultured in an in vitro environment, possess complex structures that resemble real organs, and can partially mimic the physiological functions of the tissue or organ from which they are derived. By means of organoids, researchers can deeply observe the changes of human tissues, understand the development process better, and can be used for regenerative medicine and therapeutic screening of medicines. Therefore, organoid studies have broad prospects for development.

Under specific culture conditions, organoids are healthy, capable of developing for a sufficient period of time to produce a wide range of cell types, commonly found in the human cerebral cortex. These developments have meant that brain organoids can now be used as a viable experimental system for direct investigation of diseases in patient tissues and comparison of the effects of different drugs on human brain tissue.

Brain organoids can form a variety of brain regions. They exhibit functionality and neurons that are capable of electrical excitation. They resemble human cortical development at the level of gene expression.

Neural (precursor) cell transplantation is an important means for treating neurodegenerative diseases. The main source of neural (precursor) cells is aborted fetuses, and a plurality of related clinical studies have been carried out in the united states, and clinical phase 2 has been entered into, and has therapeutic effects, and clinical trials have been terminated for ethical reasons. Human pluripotent stem cells (ES, iPSCs) are induced to differentiate in vitro into novel methods for obtaining transplanted cells. Neural cells have highly complex 3D structures, and two-dimensional planar culture cannot obtain cells with optimal functions. In 2012, american Lamaster uses human pluripotent stem cells to culture brain organoids with typical human brain tissue structures and most of the constituent cell types (ischemic vascular system and oligodendrocytes), providing an ethical bypass solution for studying human brain development.

Mesenchymal stem cells are a branch of research on stem cells, which are a class of cells having the ability to self-replicate and differentiate in multiple directions, which can constantly self-renew and under specific conditions turn into one or more cells constituting human tissues or organs. Stem cells are the cells of origin of the body and are the progenitor cells that form various tissues and organs of the human body.

However, how to select the components of the brain organoid medium and how to set the proportions of the individual components is a major challenge for those skilled in the art.

Disclosure of Invention

The invention discovers a culture medium for suspension culture of brain organoids, and by using the culture medium, a group of mesenchymal stem cells can be separated and purified from the brain organoids, and the mesenchymal stem cells have potential for the field of nerve repair.

Specifically, the technical scheme of the invention is as follows:

a culture medium for suspension culture of brain organoids, the culture medium comprising a first stage medium, a second stage medium, a third stage medium and a fourth stage medium;

the culture medium comprises NEAA1% -2%, glumax1% -2%, mercap ethanol80-120nM;

the first stage medium comprises:

GMEM basal medium, KOSR12-18%, dorsomorphin0.8-1.5 μ M, A83-010.8-1.2 μM and polyvinyl alcohol 0.5-1.5%;

the second stage medium comprises: GMEM basal medium, N2 1% -2%, SB-4315420.8-1.2 μ M, CHIR-99021 0.8-1.2 μΜ, polyvinyl alcohol 0.8-1.5%;

the third stage medium comprises: MEM basal medium, N2 1% -2%, SB-4315420.8-1.2 mu M, CHIR-99021 0.8-1.2 mu M, martigel 0.8-1.5%, polyvinyl alcohol 0.8-1.5%;

the fourth stage medium comprises: GMEM basal medium, N2 1% -2%, B27% -3%, insulin 2 μg/mL.

SB 431542 is a potent small molecule inhibitor, selectively inhibiting transforming growth factor β (TGF- β) type I receptor activin receptor-like kinase ALK5 (ic50=94 nM), ALK4 (ic50=140 nM) and ALK7. There is no inhibitory activity on other ALK family branching members such as ALK2, ALK3 or ALK6 that bind BMP proteins. SB 431542 inhibits TGF- β induced proliferation of osteosarcoma cells and promotes proliferation, differentiation and lamellar formation of endothelial cells derived from Embryonic Stem Cells (ESCs). By specifically blocking ALK/Smad signaling pathways, SB 431542 is able to maintain not only self-renewal and embryoid body formation in human embryonic stem cells, but also pluripotent stem cells. SB 431542 induces DC cell maturation in vitro while exhibiting anti-tumor activity in vivo. Patients with immune tolerance associated with tgfβ activity may activate an anti-tumor immune response using SB 431542.

CHIR-99021 (CT 99021) is a potent and selective GSK-3 alpha/beta inhibitor with IC50 of 10nM and 6.7nM. CHIR-99021 has selectivity to GSK-3 over CDC2, ERK2 and other protein kinases by more than 500 times. CHIR-99021 is also a potent Wnt/β -catenin signaling pathway activator. CHIR-99021 enhances self-renewal of mouse and human embryonic stem cells. CHIR-99021 can induce autophagy (autophagy).

Preferably, the medium comprises 1% polyvinyl alcohol.

The second aspect of the invention discloses a method for preparing mesenchymal stem cells by using the culture medium, which comprises the following steps:

s1: suspending and culturing the human pluripotent stem cells by adopting a first-stage culture medium;

s2: sequentially replacing the culture medium with a second-stage culture medium, a third-stage culture medium and a fourth-stage culture medium in the steps of D4-D6, D7-D14 and D15-D21;

s3: transferring the cells into a micro organoid bioreactor, culturing for 3-4 days, inoculating into a gelatin coated culture container, and performing wall-attached culture by adopting an N-MSCs first-stage culture medium to obtain mesenchymal stem cells.

Preferably, the first stage medium includes Y-27632 therein, and the concentration of Y-27632 is 8-12. Mu.M.

Preferably, the method further comprises step S4: and when the confluence rate of the mesenchymal stem cells in the S3 is 70-80%, adopting a serum-free MSCs culture medium for subculturing.

In some embodiments of the invention, in S1, hPSCs are cultured in conventional manner in 35mm dishes, the fluid is changed daily, the time of changing fluid is gradually advanced, and the medium is gradually increased from the initial 2ml to 3ml. When the culture was carried out at a density of 90%, the obtained human pluripotent stem cells were digested and designated day 0.

In some embodiments of the invention, the hPSCs enzyme-free digest is 0.5mM EDTA PBS.

Preferably, Y-27632 is added to the first stage medium.

More preferably, the concentration of Y-27632 is 8-12. Mu.M.

In some embodiments of the invention, in S4, the digestive juice is Ackutase or collagenase type 1 (1 mg/ml).

The subsequent passaging digestive enzyme of mesenchymal stem cells was recombinant pancreatin (0.05% pancreatin in DHanks).

In some embodiments of the invention, in S4, subculturing is performed when the adherent cells are 70% confluent, using commercial serum-free MSCs medium, at which time the cultured N-MSCs are designated as P1 generation. At the P3 generation, cells were collected for MSCs-related surface marker identification and multipotent differentiation potential identification.

In some embodiments of the invention, the serum-free MSCs medium is LONZA 12-725FultraCULTURE serum-free medium or the ncTarget medium of Anhui, inc. under the accession number RP 01020.

In a third aspect, the invention discloses mesenchymal stem cells obtained by the method.

In a fourth aspect, the invention discloses a medicament comprising mesenchymal stem cells.

Preferably, the medicament is a medicament for treating Alzheimer's disease.

In a fifth aspect, the invention discloses the use of mesenchymal stem cells according to the above in the field of neural repair. Preferably, the mesenchymal stem cells are used for preparing a medicament for treating Alzheimer's disease.

The sixth aspect of the invention discloses the use of a culture medium according to the above for the preparation of mesenchymal stem cells.

Compared with the prior art, the invention has the following beneficial effects:

the invention discloses a culture medium for suspension culture of brain organoids, which can be used for preparing and separating mesenchymal stem cells, and the early-stage pluripotent stem cells adopt a suspension culture method, so that the method has the advantages of small cell damage and high yield, polyvinyl alcohol is initially added into the later-stage cell culture medium, the shearing force of the culture is changed, the early-stage induced differentiation condition is adapted to the shearing force of suspension culture, and the use of induction factors can be reduced. The obtained mesenchymal stem cells have application in preparing medicaments in the field of nerve repair, and particularly have wide application prospects in preparing medicaments for treating Alzheimer's disease.

Drawings

Fig. 1 is a microscopic schematic diagram of a prepared mesenchymal stem cell (A: a morphological diagram of the mesenchymal stem cell under a microscope, B: a schematic diagram of mesenchymal stem cell adipogenic differentiation, C: a schematic diagram of mesenchymal stem cell osteogenic differentiation, D: a schematic diagram of mesenchymal stem cell chondrogenic differentiation).

FIG. 2 is a graph showing the flow positive results of the prepared mesenchymal stem cells;

FIG. 3 is a graph showing the flow negative results of the prepared mesenchymal stem cells;

FIG. 4 is a graph of the results of a mouse behavioural experiment-open field experiment-of mesenchymal stem cells implanted-Centimeter: cm; total Distance: total Distance.

FIG. 5 is a graph of the results of a mouse behavioural experiment-open field experiment with mesenchymal stem cells implanted (Entries in central zone: number of central zone entries; time in central zone: time in central zone; distance in central zone: distance in central zone; injection amountof: depression).

FIG. 6 is a diagram of a new Object recognition of a mouse behavioural experiment in which mesenchymal stem cells were implanted.

FIG. 7 is a graph of the behavioural experiments of mice implanted with mesenchymal stem cells-conditioned fear (continuous fear: hippocampal dependency; feed fear: amygdala dependency).

FIG. 8 is a diagram of the experimental behavioural of mice implanted with mesenchymal stem cells-Barns maze-one (escape latency).

FIG. 9 is a mouse behavioural experiment-Barns maze pattern two (Latency 1st Entrance to Target: delay first entry target) of mesenchymal stem cells.

FIG. 10 is a diagram of the mouse behavioural experiment in which mesenchymal stem cells were implanted-Barns maze pattern three (Latency 1st Entrance to Target: delay first entry into the target.

FIG. 11 is a graph showing the results of sequencing differential gene expression in different mesenchymal stem cells.

FIG. 12 is a flow chart of a length calculation of a nascent neuron;

FIG. 13 is a schematic of neurons under a confocal microscope;

FIG. 14 is a graph of Neuron Length (Neuron Length) for different groups of mesenchymal stem cells.

FIG. 15 is a graph of mouse brain slice-neuron complexity (Intersection Number: segment number).

Fig. 16 is a schematic diagram of a confocal microscope of brain sections of mice.

FIG. 17 is a graph of mouse brain slice-spin morphometric (Thin: spindle: stubby: dumbbell, mushroom: mushroom).

FIG. 18 is a graph of brain section-number of neural stem/progenitor cells in mice (SOX2+: SOX2 single positive; SOX2+GFAP+: SOX2 and GFAP co-positive).

FIG. 19 is a graph of mouse brain slice-microglial morphometric analysis (Iba-1positive cell:Iba-1 positive cells; intersection Number: segment number).

FIG. 20 is a graph of mouse brain slice-microglial morphometric analysis two (Average soma size: average body size; branch length; branch: branching).

FIG. 21 is a graph comparing the absence of added polyvinyl alcohol and the addition of 1% polyvinyl alcohol during brain organoids.

FIG. 22 shows expression of surface markers in brain organoids after addition of polyvinyl alcohol.

FIG. 23 shows brain organoids at various culture stages after addition of polyvinyl alcohol.

Detailed Description

The present application is further illustrated by the following detailed examples, it being understood that the following examples are for purposes of illustration only and are not limiting of the invention.

The instruments, devices, reagents used in the examples may be obtained from a variety of sources, such as purchased, or may be prepared.

Example 1

The embodiment discloses a culture medium for suspension culture of brain organoids, which comprises a first-stage culture medium, a second-stage culture medium, a third-stage culture medium and a fourth-stage culture medium; the following formulation percentages in the medium refer to volume ratios.

The formulation of the first stage medium is as follows:

GMEM basal medium, NEAA1%, glumax1%, mercap ethanol100nM, KOSR 15%, dorsomophin 1 μ M, A-01 μM and polyvinyl alcohol 1%.

The preparation method of the first-stage culture medium comprises the following steps: NEAA1%, glumax1%, mercap ethanol100nM, KOSR 15%, dorsomophin 1. Mu. M, A83-01. Mu.M and polyvinyl alcohol 1% were added to the GMEM basal medium in one portion.

The second stage medium comprises: GMEM basal medium, NEAA1%, glumax1%, mercapethanol 100nM, N2 1%, SB-431542 1. Mu. M, CHIR-99021 1. Mu.M, polyvinyl alcohol 1%.

The preparation method of the second-stage culture medium comprises the following steps: NEAA1%, glumax1%, mercap ethanol100nM, N2 1%, SB-431542 1 μ M, CHIR-990211 μM, polyvinyl alcohol 1% were added sequentially to the GMEM basal medium.

The third stage medium comprises: MEM basal medium, NEAA1%, glumax1%, mercap ethanol100nM, N2 1%, SB-431542 1 μ M, CHIR-990211 μ M, martigel 1%, polyvinyl alcohol 1%.

The preparation method of the third-stage culture medium comprises the following steps: NEAA1%, glumax1%, mercapethanol 100nM, N2 1%, SB-431542 1. Mu. M, CHIR-990211. Mu. M, martigel 1%, polyvinyl alcohol 1% were added sequentially to MEM basal medium.

The fourth stage medium comprises: GMEM basal medium, NEAA1%, glumax1%, mercap ethanol100nM, N2 1%, B27%, insulin 2 μg/mL.

The preparation method of the fourth-stage culture medium comprises the following steps: NEAA1%, glumax1%, mercap ethanol100nM, N2 1%, B27%, insulin 2 μg/mL were added sequentially to the GMEM basal medium.

Wherein the product model is respectively:

NEAAGibco 11140050；

GlutaMAX Gibco 35050079；

N2 Gibco A1370701；

B27 Gibco 12587010；

SB-431542MCE HY-10431；

CHIR-99021MCE HY-10182

Martigel Corning 354277

polyvinyl alcohol Sigma-Aldrich, P8136-250G

Insulin Sigma-Aldrich I9278-5ML.

Example 2

The embodiment discloses a method for preparing mesenchymal stem cells, which specifically comprises the following steps:

1. hPSCs were cultured in 35mm dishes according to conventional culture, with daily changing of the liquid, with the changing time gradually advancing, and the medium gradually increasing from the initial 2ml to 3ml. When the culture was carried out at a density of 90%, the culture was designated as day 0.

2. DPBS was used to wash the cells twice, 1 ml/dish of hPSCs enzyme-free digest was added, and the cells were placed in a 37℃incubator for 8 minutes. 3ml of ice-cold brain organoid 1 phase medium was prepared and Y-27632 was added to a final concentration of 10. Mu.M.

3. Carefully aspirate hPSCs without enzyme digest, add 1ml of ice cold brain organoid 1 stage medium containing Y-27632, shake the dish quickly left and right to shed hPSCs, transfer to one well of a common 6-well plate with a Pasteur pipette (the opening must be large, pipette tips cannot be used), and add Y-27632 of ice cold brain organoid 1 stage medium to 3ml.

4. The 6-well plate was placed in an incubator horizontal shaker (amplitude 24 mm), and incubated overnight at 60 RPM.

5. day1, microscopic cell spheres, approximately 250 μm to 350 μm in diameter. The number is about 600. Carefully transferring the culture to a 15ml centrifuge tube by using a Pasteur pipette, naturally settling for 3 minutes, taking about 2.5ml of clear solution into a new 15ml centrifuge tube, centrifuging at 3000RPM for 5 minutes, removing single cells and cell fragments, adding the single cells and cell fragments into settled cell pellets, adding fresh normal temperature brain organoids 1-stage culture medium to 9ml, carefully mixing, and sub-packaging into the wells of 3 6-well plates, wherein each well contains about 200 cell pellets and 3ml of culture medium. The culture was continued in a incubator horizontal shaker (amplitude 24 mm) at 60 RPM.

6. day2, day3, semi-liquid exchange, brain organoid 1 stage medium, incubator horizontal shaker (amplitude 24 mm), 60RPM culture.

7. day4-day6, semi-liquid exchange, and exchange into brain organoid 2 stage medium. Incubator horizontal shaker (amplitude 24 mm), 60RPM culture.

8. day7-day14, half-changed every day, changed into brain organoid 3 stage medium, incubator horizontal shaker (amplitude 24 mm), 60RPM culture.

9. day15-day21, half a day of changing liquid, changing into brain organoid 4 stage medium, transferring into micro organoid bioreactor, culturing for 21 days, and adjusting the number of each hole to 20. Micro organoid bioreactors are disclosed in patent application nos. 2017212099229, 2017212027930 or 2017212119025.

10. Cultures were removed at day22-day25, settled and digested with accutase for 15min before being blown into single cells, and half-changed every 3 days in flasks coated with N-MSCs first-stage medium gelatin. In the second half-change, the suspension cells are removed.

11. When the adherent cells are 70% confluent, subculturing is performed, and commercial serum-free MSCs culture medium is used, wherein the cultured N-MSCs are recorded as P1 generation, and the schematic diagram under a microscope of cells 2 is shown in FIG. 1. The serum-free MSCs culture medium is LONZA 12-725FultraCULTURE serum-free culture medium or ncTarget culture medium of the stock name RP01020 of the company of the Ministry of Biotechnology of Anhui.

12. At the P3 generation, the collected cells are subjected to MSCs related surface marker identification and multi-directional differentiation potential identification, and a schematic diagram under a cell microscope is shown in figure 1.

The mesenchymal stem cells of the generation P3 were then subjected to flow assay, and the obtained cells were further verified to be mesenchymal stem cells, and the results are shown in FIGS. 2 to 3.

In this example, a control group was also provided, the only difference between the control group and the above method being that no polyvinyl alcohol was added to the first stage medium formulation. The control group found no way to grow organoids.

Example 3

This example investigated the effect of mesenchymal stem cells on the nerve repair function of mice. This example uses AD model (alzheimer's disease) mice (double transgenes, human presenilins and aβ) for treatment efficacy evaluation, and the experiments are divided into four groups:

PBS group: a control group;

MSC group: umbilical cord derived MSCs;

TC group: another subject, genetically modified MSCs, over-express TIMP 2N-MSCs in human umbilical MSCs using AAV; N-MSC group: the brain-derived MSCs, i.e. the mesenchymal stem cells prepared in example 2.

The experimental design is as follows:

experiment design: and randomly grouping 9 mice in each group, wherein three mice are selected to be used as GFP retrovirus markers of DG proliferation-stage neural stem cells, and the method is mainly used for evaluating the development of new-born neurons and the integration capacity of neural networks after MSCs are analyzed in the future.

Treatment: the treatment period was 8 weeks, the tail vein injection was carried out once a week, and the cell group dose was 10 ⁵ Cells/mice. After treatment, the 9w is used for carrying out a mine field experiment and a new object identification experiment; the 10 th w-12w is used for carrying out a Barns maze experiment; conditioned fear experiments were performed at 13 w.

After the mice are sacrificed, the whole viscera are checked, and no obvious tumor is generated; the mice were fixed by 6 infusions per group, 3 direct liquid nitrogen frozen stock and hippocampal tissues were taken for sequencing.

The specific test results are as follows:

1. the mine experiments are carried out on four groups of mice, the experimental results are shown in figures 4-5, and the four groups of mice have similar activity degrees, and the mice in the N-MSC treatment group are more active and more general.

2. New object recognition experiments were performed on four groups of mice, scoring% = new object explored time/new object explored time + old object explored time. The results are shown in FIG. 6, and it can be seen from the graph that the treatment group has higher score for new object recognition and the N-MSC treated group mice have better short-term memory.

3. The four groups of mice were subjected to a conditional fear test, and the contextual fear of the N-MSC treated group was found to be significantly improved, while the fear test with clue cues was not significantly different, and the test results are shown in FIG. 7.

4. The barnes maze test was performed on four groups of mice, and a significant difference was found in the first training time after the adaptation period, indicating that the N-MSC treated group of mice had a greater learning ability, and the results are shown in fig. 8.

The fourth day training results showed that all mice could find the target hole within 30 seconds, indicating that training was successful and available for testing, and the results are shown in fig. 9.

The test results on the fifth day show no obvious difference, which indicates that the short-time memories of the four groups of mice are similar; the results on day 12 show that the long term memory of the mice in the N-MSC treated group is improved and the results are shown in FIG. 10.

5. Differential gene expression sequencing was performed on the genes of four groups of mice, and the results are shown in fig. 11.

6. The flow chart of the brain slice-neuron length analysis of the four groups of mice is shown in fig. 12, and the result schematic diagrams are shown in fig. 13-14.

Further studies on the complexity of neurons, the results are shown in FIG. 15, where the N-MSC group mouse neurons were longer and the more branched differences were statistically significant.

7. Four groups of mice were studied for neuron terminal axons (spines), and confocal microscopy was used to photograph the terminal 5 μm of the newly generated neurons, and the spines were counted using a Neurolucida classification, as shown in FIGS. 16-17, in which Thin refers to spindle type, stubby refers to dumbbell type, and Mushroom refers to Mushroom type. As can be seen from the figure, the total number of spines in the mice of the N-MSC group is the greatest, and the neuron axons are more mature.

8. The number of neural stem/progenitor cells in brain sections of four groups of mice was studied and the results are shown in fig. 18. The number of the treated mouse neural stem cells (common yang) is more, which indicates that MSC has stronger effect of maintaining dryness.

9. The results of analysis of microglial cell morphology in brain sections of four groups of mice are shown in fig. 19, and after treatment, the number of microglial cells in mice DG and CA1 regions Iba-1 is reduced without obvious difference, and the difference is statistically significant.

Further studies, as shown in figure 20, found that the N-MSC treated group mice had smaller microglial cell bodies, fewer branches and shorter branch lengths. FIG. 21 is a graph comparing the absence of added polyvinyl alcohol and the addition of 1% polyvinyl alcohol during brain organoids. FIG. 22 shows expression of surface markers in brain organoids after addition of polyvinyl alcohol. FIG. 23 shows brain organoids at various culture stages after addition of polyvinyl alcohol.

From the above, the mesenchymal stem cells prepared by the method have potential for nerve repair, and can be possibly used in medicines for treating Alzheimer's disease in future.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (3)

1. A culture medium for suspension culture of brain organoids is characterized in that,

the culture medium comprises a first stage culture medium, a second stage culture medium, a third stage culture medium and a fourth stage culture medium;

the first-stage culture medium, the second-stage culture medium, the third-stage culture medium and the fourth-stage culture medium comprise NEAA1% -2%, glumax1% -2% and Mercap ethanol80-120nM;

the first stage medium comprises: GMEM basal medium, KOSR12-18%, dorsomorphin0.8-1.5 mu M, A-01.8-1.2 mu M and polyvinyl alcohol 0.5-1.5%;

the second stage medium comprises: GMEM basal medium, N2 1% -2%, SB-4315420.8-1.2 μ M, CHIR-99021 0.8-1.2 μΜ, polyvinyl alcohol 0.8-1.5%;

the third stage medium comprises: MEM basal medium, N2 1% -2%, SB-4315420.8-1.2 mu M, CHIR-99021 0.8-1.2 mu M, martigel 0.8-1.5% and polyvinyl alcohol 0.8-1.5%;

the fourth stage medium comprises: GMEM basal medium, N2 1% -2%, B27% -3%, insulin 2 μg/mL.

2. The medium of claim 1, wherein the first stage medium, second stage medium, and third stage medium comprise 1% polyvinyl alcohol.

3. Use of a culture medium according to any one of claims 1-2 for the preparation of mesenchymal stem cells.

Images