CN115109801A

CN115109801A - Slow virus transfection transfer aid and application thereof

Info

Publication number: CN115109801A
Application number: CN202211023688.6A
Authority: CN
Inventors: 谢海涛; 方晓; 马丽雅
Original assignee: Shenzhen Xiankangda Life Science Co ltd
Current assignee: Shenzhen Xiankangda Life Science Co ltd
Priority date: 2022-08-25
Filing date: 2022-08-25
Publication date: 2022-09-27
Anticipated expiration: 2042-08-25
Also published as: CN115109801B; WO2024040980A1

Abstract

The invention discloses a lentivirus transfection transfer aid and application thereof; the transfer aid comprises a serum-free culture medium containing 1 mg/ml-50 mg/ml of basic amino acid. The transfection transfer aid can effectively neutralize electrostatic repulsion between sialic acid on the cell surface and virus particles, promotes adsorption, and enables lentiviruses to easily enter immune cells, so that the expression of chimeric antigen receptors, such as the positive rate of CAR-T cells, is effectively improved, has no cytotoxicity, and is simple to operate by adopting the lentivirus transfection process of the transfer aid, and residual removal operation is not required after transfection is finished, so that the operation of the transfection process is simpler and more convenient.

Description

Lentivirus transfection transfer aid and application thereof

Technical Field

The invention relates to the technical field of lentivirus transfection, in particular to a lentivirus transfection transfer promoter and application thereof.

Background

With the development of the immunotherapy industry, CAR-T cell products come into the market, and more research institutions and pharmaceutical companies are invested in CAR-T research and development, clinical trials, and industrial preparation. CAR-T cells are by far the most popular study. CAR-T therapy, i.e., Chimeric Antigen Receptor T-Cell Immunotherapy, is one of adoptive Cell Immunotherapy (ACT), and its family also includes methods such as TIL (tumor infiltrating lymphocyte therapy), TCR-T (engineered T-Cell Receptor therapy), CAR-NK, etc. Chimeric antigen receptor T cells (CAR-T cells) are those in which the antigen binding portion of an antibody that recognizes a tumor antigen is coupled in vitro to the intracellular portion of the CD 3-zeta chain or fcsri γ to form a chimeric protein, and the Chimeric Antigen Receptor (CAR) is expressed by transfecting the T cells of a patient by gene transduction.

Lentiviruses are the best method for gene transduction, have the advantages of high transduction efficiency, integration of T cell genome and continuous expression of target proteins, and are incomparable with transfection, electrotransformation, retroviruses, adenoviruses and the like. However, the entry of lentiviruses carrying the gene of interest into T cells is hindered due to the characteristics of the lentiviruses themselves and the T cells themselves, and the positive rate of CAR-T cells cannot be guaranteed.

At present, most of commercially available lentivirus transfection transfer aids take Polybrene (Polybrene) as a main component, which can improve the lentivirus transfection efficiency and the positive rate of CAR-T cells. Polybrene is a polycationic polymer commonly used in DNA transfection experiments of mammalian cells to enhance the transfection efficiency of liposomes; polybrene is currently widely used for retrovirus-mediated gene transfection, lentivirus-mediated gene transfection, probably by neutralizing electrostatic repulsion between cell surface sialic acid and virus particles to promote adsorption. Thereby leading the virus to more easily bring the carried target gene into transfected cells and improving the transfection effect. However, Polybrene is expensive and toxic to some cells (such as terminally differentiated neurons, DC cells, T cells, etc.), and affects the expansion and differentiation of cells; since polybene is a polymer additive, its residue needs to be strictly controlled. The preparation cost is increased, and the research and development difficulty is increased.

Disclosure of Invention

Based on the problems, the invention provides a slow virus transfection aid and a slow virus transfection aid transfection method which are low in cost, low in toxicity and free of residual control.

The first technical scheme of the invention is as follows:

a lentivirus transfection transfer aid comprises a serum-free culture medium containing basic amino acid with the concentration of 1 mg/ml-50 mg/ml.

In one embodiment, the basic amino acids include 1mg/ml to 50mg/ml histidine, 1mg/ml to 50mg/ml lysine, and 1mg/ml to 50mg/ml arginine in the lentivirus transfection facilitator.

In one embodiment, the serum-free medium in the lentivirus transfection adjuvant is any one of DMEM, IMEM, RPMI-1640, KPM-581 and OPTMIZER TCELL EXPANSFCTS.

The second technical scheme of the invention is as follows:

a lentivirus transfection-assisted transfection method comprises the following steps:

separating immune cells from peripheral blood;

suspending the immune cells by adopting a serum-free culture medium, culturing, activating the immune cells, and activating to obtain an immune cell suspension;

adding the lentivirus transfection transfer aid into the activated immune cell suspension to obtain immune cell transfection suspension;

adding lentivirus into the immune cell transfection suspension according to the MOI amount to perform lentivirus transfection;

after the lentivirus transfection is completed, a complete culture medium is added to culture the transfected immune cells.

In one embodiment, the serum-free medium is added at a rate of 1.0 × 10 per each immune cell resuspension and activation step ⁶ Adding 350 μ l of each cell; the activation time does not exceed 24 hours.

In one embodiment, in the step of preparing the immune cell transfection suspension, 5 to 30 μ l of a transfer promoter is added to 1ml of cell suspension.

In one embodiment, in the step of transfecting lentivirus, the lentivirus is added in an amount of MOI = 1-MOI =10, and the transfection time is 6-24 hours.

In one embodiment, the complete medium is added in an amount to maintain the cell concentration at 1.0X 10 in the step of culturing the transfected immune cells ⁶ Individual cells/ml.

In one embodiment, the immune cells are T cells, and may be NK, CIK, NKT, or other cells. In the immune cell sorting step, the immune cells are preferably T cells. If the T cell sorting is positive sorting, activating magnetic beads to sort the T cells by adopting CD3/CD28beads sorting; if negative selection is used for T cell sorting, the resulting T cells are also activated with CD3/CD28 beads.

Compared with the prior art, the invention also has the following advantages:

1) the transfer aid mainly comprises basic amino acid, and the added amino acid does not carry other residual components, so that the transfer aid has no toxic effect on cells, can improve the activity of the cells, reduces the toxic and side effects of lentiviruses on the cells, and ensures that immune cells transfected by the lentiviruses have higher clinical safety;

2) the basic amino acid takes histidine, lysine and arginine as main components, has wide market sources and low price, and ensures that the manufacturing cost of the lentivirus transfection process is low;

3) histidine, lysine and arginine in the basic amino acid can neutralize charges on immune cells and have the function of repelling static electricity; meanwhile, essential amino acid can be provided for cell growth;

4) the lentivirus transfection process using the basic amino acid as the transfer assisting agent is simple to operate, and residue removal operation is not needed after transfection is completed, so that the transfection process is simpler and more convenient to operate.

Drawings

FIG. 1 is a flow chart of a process for transfecting an immune cell with a lentivirus provided by the present invention;

FIG. 2 is a graph of the positive rate of CAR-T after lentivirus transfection of T cells in example 6;

FIG. 3 is a graph of the detection of the positive rate of CAR-T after transfection of T cells with lentivirus in example 7;

FIG. 4 is a graph of the positive rate of CAR-T after lentivirus transfection of T cells in example 8;

FIG. 5 is a graph of the detection of the positive rate of CAR-T after lentivirus transfection of T cells in example 9;

FIG. 6 is a graph of the positive rate of CAR-T after lentivirus transfection of T cells in example 10;

FIG. 7 is a graph of the detection of the positive rate of CAR-T after transfection of T cells with lentivirus in example 11;

FIG. 8 is a graph of the detection of the positive rate of CAR-T after transfection of T cells with lentivirus in example 12;

FIG. 9 is a graph of the detection of the positive rate of CAR-T after transfection of T cells with lentivirus in example 13;

FIG. 10 is a graph of the positive rate of CAR-T after lentivirus transfection of T cells in example 14;

FIG. 11 is a graph of the detection of the positive rate of CAR-T after transfection of T cells with lentivirus in example 15;

FIG. 12 is a graph showing the detection of the positive rate of the NK cell negative control in example 15;

FIG. 13 is a graph showing growth expansion of lentivirus-transfected T cells of examples 11 to 14.

Detailed Description

The preferred embodiments of the present invention will be described in further detail with reference to the accompanying drawings.

Specifically, the components of the lentivirus transfection transfer aid provided by the invention comprise a serum-free culture medium containing 1 mg/ml-50 mg/ml basic amino acid. The transfection transfer aid can effectively neutralize electrostatic repulsion between cell surface sialic acid and virus particles, promote adsorption, and enable lentiviruses to easily enter immune cells, so that the expression of chimeric antigen receptors, such as the positive rate of CAR-T cells, is effectively improved, and the transfer aid has no cytotoxicity.

Further, the serum-free medium may be any of serum-free media such as DMEM, IMEM, RPMI-1640, KPM-581, OPTMIZER TCELL EXPANSFCTS, etc. For the selection of the serum-free culture medium, a scientific research-grade culture medium can be selected in the research and development stage, and a clinical-grade culture medium can be selected in the clinical stage;

further, the basic amino acids include histidine, lysine and arginine.

As shown in FIG. 1, the invention also provides a process for transfecting lentivirus by using the transfer promoter, which comprises the following steps;

s1, collecting human Peripheral Blood (PBMC), and sorting immune cells;

s2, transferring the selected immune cells to a culture bottle for culture after being resuspended by a serum-free culture medium, and activating the immune cells at the same time, thereby obtaining activated immune cell suspension;

s3, adding the transfection transfer aid into the activated immune cell suspension to obtain immune cell transfection suspension;

s4, adding lentivirus into the immune cell transfection suspension according to the MOI amount to carry out lentivirus transfection;

and S5, after the lentivirus transfection is finished, transferring the lentivirus into a culture bottle, adding a complete culture medium, and culturing the transfected immune cells.

The immune cell culture may be a culture flask, a culture bag or a culture dish.

Further, in step S1, when immune cells are sorted from PBMCs, positive selection or negative selection may be selected; if the immune cells are T cells and positive selection is adopted, selecting CD3/CD28beads from the PBMCs to select the T cells by selecting activated magnetic beads, carrying out negative selection on the T cells by adopting a negative selection kit, and adding CD3/CD28beads magnetic beads into the obtained T cells to activate the T cells.

Further, in step S2, the serum-free medium is added in an amount of 1.0X 10 ⁶ The cells were added in 350. mu.l and the time for immune cell activation did not exceed 24 hours.

Further, in the step S3, the addition amount of the transfer aid is 5-30 mul added to 1ml of cell suspension; preferably, 10. mu.l of the transfer aid is added to 1ml of the cell suspension.

Further, in step S4, the lentivirus is added in an amount of MOI = 1-MOI =10, and the transfection time is 6-24 hours; the optimal transfection time is 6 hours; when the immune cells are cells such as CIK, NK, NKT, DC, etc., the amount of lentivirus to be added may be at most MOI = 100.

Further, in step S5, the lentivirus is cultured after transfection, and the amount of the complete medium added is 1.0X 10 ⁶ Individual cells/mL operation; the complete medium contained 10 v/v% FBS.

The immune cells used in the present invention are T cells, CIK cells, NK cells, NKT cells, DC cells, and the like.

T cells and NK cells are exemplified below, and are described in further detail by specific examples.

First, preparation of transforming aid for slow virus transfection

Example 1

Calculated as the concentration of amino acids in the solution, the ingredients are provided: histidine with the concentration of 10mg/ml, lysine with the concentration of 10mg/ml, arginine with the concentration of 10mg/ml and DMEM serum-free culture medium as the rest are mixed to prepare the lentivirus transfection transfer aid A.

Example 2

Calculated as the concentration of amino acids in the solution, the ingredients are provided: histidine at a concentration of 50mg/ml, lysine at a concentration of 10mg/ml, arginine at a concentration of 10mg/ml, and the balance OPTMIZER TCELL EXPANSFCTS serum-free medium were mixed to prepare a lentivirus transfection adjuvant B.

Example 3

Calculated as the concentration of amino acids in the solution, the ingredients are provided: histidine with the concentration of 25mg/ml, arginine with the concentration of 1mg/ml, arginine with the concentration of 10mg/ml and RPMI-1640 serum-free culture medium as the rest are mixed to prepare the lentivirus transfection transfer aid C.

Example 4

Calculated as the concentration of amino acids in the solution, the ingredients are provided: histidine with the concentration of 1mg/ml, lysine with the concentration of 10mg/ml, arginine with the concentration of 50mg/ml and IMEM serum-free culture medium for the rest are mixed to prepare the lentivirus transfection adjuvant D.

Example 5

Calculated as the concentration of amino acids in the solution, the ingredients are provided: histidine with the concentration of 1mg/ml, lysine with the concentration of 1mg/ml, arginine with the concentration of 1mg/ml and KPM-581 serum-free medium for the rest to prepare the lentivirus transfection transfer assistant E.

Second, lentivirus transfection of immune cells

Example 6

T cells were sorted from PBMC using CD3/CD28beads activated magnetic beads, and 1.0X 10 cells were collected ⁶ The T cell of (1);

resuspend 1.0X 10 with 350. mu.l physiological saline ⁶ (ii) T cells, which are activated simultaneously;

adding lentivirus according to MOI =3, and performing transfection treatment;

after 6 hours of transfection, the ratio was 1.0X 10 ⁶ Adding 1ml of complete culture medium into each ml of cells, and culturing the transfected T cells;

the 3 rd day of cultureNumber of T cells, in a 0.5X 10 ⁶ Cells/ml were replenished and the positive rate of CAR flow assay on day 5 was 14.97%, as shown in figure 2.

Example 7

Sorting activated beads from PBMC with CD3/CD28beads sorting activated beads T cells were sorted out and taken at 1.0X 10 ⁶ The T cell of (1);

3.5ul of transfer aid B prepared in example 2 was added to the T cell suspension;

adding lentivirus according to MOI =5, and performing transfection treatment;

20 hours after transfection, at 1.0X 10 ⁶ Adding 1ml of complete culture medium into each ml of cells, and culturing the transfected T cells;

the number of the culture day 3 was counted at 0.5X 10 ⁶ Cells/ml were replenished and the positive rate of flow-tested CAR on day 5 was 47.40%, as shown in figure 3.

Example 8

T cells were sorted from PBMCs using CD3/CD28beads activated magnetic beads, 1.0X 10 ⁶ The T cell of (1);

to the T cell suspension, 3.5ul Polybrene was added;

adding lentivirus according to MOI =8, and performing transfection treatment;

the number of the culture day 3 was counted at 0.5X 10 ⁶ Cells/ml were replenished and the positive rate of flow-tested CAR on day 5 was 50.03%, as shown in figure 4.

Example 9

resuspension of 1.0X 10 with 350. mu.l of KPM-581 serum-free medium ⁶ (ii) T cells, which are activated simultaneously;

to the T cell suspension, 1.75ul of the transfer aid E prepared in example 5 was added;

adding lentivirus according to MOI =10, and performing lentivirus transfection treatment;

24 hours after transfection, at 1.0X 10 ⁶ Adding 1ml of complete culture medium into each ml of cells, and culturing the transfected T cells;

the number of the culture day 3 was counted at 0.5X 10 ⁶ Cells/ml were replenished and the positive rate of flow-tested CAR on day 5 was 28.23%, as shown in figure 5.

Example 10

resuspension of 1.0X 10 with 350. mu.l IMEM serum-free medium ⁶ (ii) T cells, which are activated simultaneously;

to the T cell suspension, 6.5ul of transfer aid D prepared in example 4 was added;

adding lentivirus according to MOI =1, and performing lentivirus transfection treatment;

15 hours after transfection, at 1.0X 10 ⁶ Adding 1ml of complete culture medium into each ml of cells, and culturing the transfected T cells;

the number of the culture day 3 was counted at 0.5X 10 ⁶ Cells/ml were replenished and the CAR was flow tested on day 5 with a positive rate of 58.67%, as shown in figure 6.

Example 11

resuspending 1.0X 10 in 350. mu.l RPMI-1640 serum-free Medium ⁶ (ii) T cells, which are activated simultaneously;

4.5ul of the transfer aid C from example C was added to the T cell suspension;

adding lentivirus according to MOI =7, and performing transfection treatment on the lentivirus;

12 hours after transfection, the ratio was 1.0X 10 ⁶ Adding 1ml of complete culture medium into each ml of cells, and culturing the transfected T cells;

the number of the culture day 3 was counted at 0.5X 10 ⁶ Cells/ml fluid was replenished, and the positive rate of flow-assay CAR on day 5 was 48.23%, as shown in figure 7;

then counting every 2 days, supplementing liquid, culturing till the 13 th day, the cells are 1.0X 10 ⁶ The cells/ml were expanded to 6.7X 10 ⁸ Cell/ml, 670 fold amplification, as shown in table 2 and figure 13.

Example 12

resuspension of 1.0X 10 with 350. mu.l of OPTMIZER TCELL EXPANSFCTS serum-free Medium ⁶ (ii) T cells, which are activated simultaneously;

to the T cell suspension, 10.5ul of the transfer aid B prepared in example 2 was added;

adding lentivirus according to MOI =6, and performing transfection on the lentivirus;

8 hours after transfection, at 1.0X 10 ⁶ Adding complete culture medium into cells/ml, and culturing the transfected T cells;

the number of the culture day 3 was counted at 0.5X 10 ⁶ Cell/ml fluid replacement, flow-tested CAR positive rate 67.27% on day 5, as shown in figure 8;

then counting every 2 days, supplementing liquid, culturing till the 13 th day, the cells are 1.0X 10 ⁶ Expansion of cells/ml to 7.08X 10 ⁸ Cells/ml, expansion fold was 708 fold as shown in table 2 and fig. 13.

Example 13

resuspension of 1.0X 10 with 350. mu.l DMEM serum-free medium ⁶ (ii) T cells, which are activated simultaneously;

3.0ul of the transfer aid A prepared in example 1 was added to the T cell suspension;

adding lentivirus according to MOI =3, and performing transfection on the lentivirus;

the number of the culture day 3 was counted at 0.5X 10 ⁶ Cells/ml fluid was replenished, and the positive rate of flow-assay CAR on day 5 was 54.11%, as shown in figure 9;

then counting every 2 days, supplementing liquid, culturing till the 13 th day, the cells are 1.0X 10 ⁶ The cells/ml were expanded to 6.32X 10 ⁸ Cell/ml, up to 632 fold amplification, as shown in table 2 and figure 13.

Example 14

3.5ul of Polybrene as a transfer aid was added to the T cell suspension;

adding lentivirus according to MOI =6, and carrying out transfection on the lentivirus;

the number of the culture day 3 was counted at 0.5X 10 ⁶ Cells/ml fluid was replenished, and the positive rate of flow-assay CAR on day 5 was 69.20%, as shown in figure 10;

then counting every 2 days, supplementing liquid, culturing till the 13 th day, the cells are 1.0X 10 ⁶ The cells/ml were expanded to 5.1X 10 ⁸ Cell/ml, expansion fold 510 fold, as shown in Table 2 and FIG. 13

Example 15

NK cells were sorted from PBMC using sorting magnetic beads, and 1.0X 10 cells were collected ⁶ The NK cell of (1);

resuspension of 1.0X 10 with 350. mu.l DMEM serum-free medium ⁶ (ii) NK cells, simultaneously activating NK cells;

to the NK cell suspension, 3.5ul of the transfer aid A prepared in example 1 was added;

24 hours after transfection, at 1.0X 10 ⁶ Adding 1ml of complete culture medium into each ml of cells, and culturing the transfected NK cells;

the number of the culture day 3 was counted at 0.5X 10 ⁶ Cells/ml were replenished and the positive rate of flow-tested CAR on day 5 was 30.81%, as shown in figure 11.

Third, detection of lentivirus transfection assay

1. Positive rate detection of CAR-T and CAR-NK

1.1, examples 6, 7 and 8 above, as shown in FIGS. 2, 3, 4 and Table 1, in example 8, when lentivirus was cultured with physiological saline alone, the transduction efficiency (i.e., CAR-positive rate) of lentivirus was 14.97%; in example 7, after the lentivirus was cultured with normal saline and the transfer aid B was added, the CAR positive rate was 47.40%; example 8 is a prior lentivirus transfection procedure, which uses normal saline to culture lentivirus and adds polybrene as a transfer promoter, and the CAR positive rate is 50.03%. Thus, it is clear from examples 6, 7 and 8 that the basic amino acids in the transducible agent promote the transduction of lentiviruses with a transduction efficiency close to that of polybrene.

It is now common to use the cotransferase agent polybrene for lentivirus transfection because: in the process of transfecting T cells by lentivirus, the cationic polymer carried by polybrene can neutralize electrons in the T cells, so that the lentivirus transduction rate of the polybrene is improved.

Table 1 shows the parameters of lentivirus transfection and CAR positivity for each of examples 6 to 15

Note: the transfer aids a to E in table 1 are respectively the transfer aids formulated correspondingly in examples 1 to 5.

1.2 in examples 9 to 14 above, one of serum-free media such as DMEM, IMEM, RPMI-1640, KPM-581, OPTMIZER TCELL EXPANSFCTS, etc. was used for the lentiviral transfection aid, while the transfection aids 1 to E prepared in examples 1 to 5 of the present invention and the conventional transfection aid polybrene were used for the lentiviral transfection aid, respectively. As can be seen from fig. 2 to 10 and table 1:

1) compared with normal saline, the serum-free culture medium is beneficial to lentiviral transduction, namely the CAR positive rate is generally higher;

2) in lentivirus transduction, the transfer promoters A to E increase with the addition of the transfer promoters, so that the CAR positive rate also increases, namely the lentivirus transduction rate is also improved;

3) the transduction rate of the transfer promoter provided by the invention can reach the transduction rate of the existing transfer promoter polybrene.

1.3, example 15 using NT cell lentivirus transfection, compared to examples 6 to 14 using T cell lentivirus transfection, it can be seen from FIGS. 11 and 12 and Table 1 that NK cells also have lentivirus transfection effect under the action of the transfer promoter provided by the present invention, except that the transduction rate is relatively low, i.e. the positive rate of CAR is 30.81%.

Second, CAR-T cell expansion culture after transfection

FIG. 13 is a graph of the expansion profile of CAR-T cell culture processes following lentivirus transfection in examples 11 to 14; table 2 shows the number of cells grown in FIG. 13.

TABLE 2 statistics of CAR-T cell growth after transfection, (. times.10) ⁶ cell/mL)

As can be seen from Table 2 and FIG. 13, after transfection, CAR-T cells were subjected to static culture and cell counting on the day corresponding to the culture time, and after the 13 th day of culture, the amplification factor of CAR-T cells shows that the transfer promoter of the present invention has less toxicity to cells, and the amplification factor of cells is higher than that of polybrene added as the transfer promoter.

It should be understood that the above description is illustrative of the preferred embodiment of the present invention and is not to be considered as limiting the scope of the invention, which is defined by the appended claims.

Claims

1. A lentivirus transfection transfer aid is characterized by comprising a serum-free culture medium containing 1-50 mg/ml of basic amino acid.

2. The lentiviral transfection aid according to claim 1, wherein the basic amino acid comprises 1mg/ml to 50mg/ml histidine, 1mg/ml to 50mg/ml lysine and 1mg/ml to 50mg/ml arginine.

3. The lentiviral transfection aid according to claim 1, wherein the serum-free medium is any one of DMEM, IMEM, RPMI-1640, KPM-581 and OPTMIZER TCELL EXPANSFMCTS.

4. A lentivirus transfection-assisted transfection method is characterized by comprising the following steps:

separating immune cells from peripheral blood;

adding the transfection aid of any one of claims 1 to 3 to the activated immune cell suspension to obtain an immune cell transfection suspension;

according to the MOI amount, adding lentivirus into the immune cell transfection suspension to perform lentivirus transfection;

5. The method of claim 4, wherein the serum-free medium is added at a rate of 1.0X 10 per 1.0X 10 in the steps of resuspension and activation of the immune cells ⁶ Adding 350 μ l of each cell; the activation time does not exceed 24 hours.

6. The method of claim 4, wherein the step of preparing the immune cell transfection-assisting suspension comprises adding 5-30 μ l of the transfection-assisting agent per 1ml of the cell suspension.

7. The method of claim 4, wherein the lentivirus transfection is performed in an amount of MOI = 1-MOI =10 and the transfection time is 6-24 hours.

8. The lentivirus of claim 4A transfection-assisted method characterized in that, in the step of culturing transfected immune cells, the complete medium is added in an amount such that the cell concentration is maintained at 1.0X 10 ⁶ Individual cells/ml.

9. The method of any one of claims 4 to 8, wherein the immune cells are T cells.

10. The method of claim 9, wherein in the step of immune cell sorting, if T cell sorting is positive sorting, CD3/CD28beads sorting is used to activate magnetic beads to sort T cells; if negative selection is used for T cell sorting, the resulting T cells are also activated with CD3/CD28 beads.